Large and small t antigens of merkel cell polyomavirus, nucleic acid constructs and vaccines made therefrom, and methods of using same

ABSTRACT

Nucleic acid molecules and compositions comprising one or more nucleotide sequences that encode a consensus Merkel Cell Polyomavirus (MCV) T antigen. Immunomodulatory methods and methods of inducing an immune response against MCV are disclosed. Method of treating infection by MCV and methods of treating or preventing Merkel Cell Carcinoma associated with MCV are disclosed. Modified consensus MCV T antigens are disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application SerialNo. 62/619,161, filed Jan. 19, 2018, the contents of which areincorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention relates to vaccines for inducing immune responsesand treating individuals infected with MCV and/or treating or preventingMerkel Cell Carcinoma (MCC). The present invention relates to consensusMCV large T antigen (LTAg) and small t antigen (STAg) oncoproteins andnucleic acid molecules which encode the same.

BACKGROUND OF THE INVENTION

Merkel Cell Polyomavirus (MCV) has gained recent attention due to itslink with Merkel Cell Carcinoma (MCC), an aggressive human skin cancer.Approximately 1,500 new cases of MCC are diagnosed per year in theUnited States, and the mortality rate for subjects with MCC remains at46%. MCC kills more patients than cutaneous T cell lymphoma and chronicmyeloid leukemia. A majority (approximately 75%) of MCCs containclonally integrated viral DNA and express viral T antigen transcriptsand protein.

Currently there are no vaccines against MCC being tested in clinicaltrials. Therefore, there is need in the art for therapeutic vaccinesagainst MCV and MCC. The current invention satisfies this unmet need.

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to an immunogenic compositioncomprising a nucleic acid molecule encoding at least one modified MerkelCell Polyomavirus (MCV) T antigen, wherein the T antigen comprises atleast one mutation that disrupts at least one oncogenic feature of anative MCV T antigen. In one embodiment, the at least one oncogenicfeature is at least one of CR1 binding, DnaJ binding, phophatasepp2A-binding binding, Rb binding, ATPase activity, helicase activity,chaperone protein binding, hVam6p binding, Fbxw7 binding, originbinding, and transformation.

In one embodiment, the at least one mutation is a mutation at an aminoacid at least one of D44, W209, E216, L142, L91, K92, D93, Y94 or M95.In one embodiment, the at least one mutation is at least one of a D44Nmutation, a W209A, an E216K mutation, an L142A mutation, an L91Amutation, a K92A mutation, a D93A mutation, a Y94A mutation or a M95Amutation. In one embodiment, the modified MCV T antigen comprises atleast one of a D44N mutation, a W209A, or an E216K mutation. In oneembodiment, the modified MCV T comprises a D44N mutation, a W209A, andan E216K mutation.

In one embodiment, the at least one MCV T antigen is a large T antigen(LTAg) or a small t antigen (STAg.) In one embodiment, the at least oneMCV T antigen is a combination of a LTAg and a STAg.

In one embodiment, the nucleic acid molecule encodes a peptidecomprising an amino acid sequence of a) an amino acid sequence having atleast about 90% identity over an entire length of the amino acidsequence to at least one of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, b)an immunogenic fragment comprising at least about 90% identity over atleast 60% of the amino acid sequence to at least one of SEQ ID NO:2, SEQID NO:4 or SEQ ID NO:6, c) the amino acid sequence of SEQ ID NO:2, SEQID NO:4 or SEQ ID NO:6, or d) an immunogenic fragment comprising atleast 60% of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQID NO:6.

In one embodiment, the nucleic acid molecule is a DNA molecule or a RNAmolecule.

In one embodiment, the nucleic acid molecule comprises a nucleotidesequence at least one of a) a nucleotide sequence having at least about90% identity over an entire length of a nucleotide sequence to at leastone of SEQ ID NO: 1, SEQ ID NO:3 or SEQ ID NO:5, b) an immunogenicfragment of a nucleotide sequence having at least about 90% identityover at least 60% of the nucleotide sequence to at least one of SEQ IDNO: 1, SEQ ID NO:3 or SEQ ID NO:5, c) a nucleotide sequence of SEQ IDNO:1, SEQ ID NO:3 or SEQ ID NO:5, or d) an immunogenic fragment of anucleotide sequence of SEQ ID NO: 1, SEQ ID NO:3 or SEQ ID NO:5.

In one embodiment, the nucleotide sequence encoding the peptide isoperably linked to at least one regulatory sequence. In one embodiment,the regulatory sequence is at least one of a start codon, an IgE leadersequence or a stop codon.

In one embodiment, the nucleic acid molecule encodes a peptidecomprising an amino acid sequence of at least one of a) an amino acidsequence having at least about 90% identity over an entire length of theamino acid sequence to at least one of SEQ ID NO:2, SEQ ID NO:4 or SEQID NO:6, b) an immunogenic fragment comprising at least about 90%identity over at least 60% of the amino acid sequence to at least one ofSEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, c) the amino acid sequence ofSEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, or d) an immunogenic fragmentcomprising at least 60% of the amino acid sequence of SEQ ID NO:2, SEQID NO:4 or SEQ ID NO:6, operably linked to an amino acid sequence as setforth in SEQ ID NO:7.

In one embodiment, the nucleic acid molecule comprises a nucleotidesequence of at least one of a) a nucleotide sequence having at leastabout 90% identity over an entire length of a nucleotide sequence to atleast one of SEQ ID NO: 1, SEQ ID NO:3 or SEQ ID NO:5, b) an immunogenicfragment of a nucleotide sequence having at least about 90% identityover at least 60% of the nucleotide sequence to at least one of SEQ IDNO: 1, SEQ ID NO:3 or SEQ ID NO:5, c) a nucleotide sequence of SEQ IDNO: 1, SEQ ID NO:3 or SEQ ID NO:5, or d) an immunogenic fragment of anucleotide sequence of SEQ ID NO: 1, SEQ ID NO:3 or SEQ ID NO:5,operably linked to an nucleotide sequence encoding SEQ ID NO:7.

In one embodiment, the nucleic acid molecule comprises an expressionvector.

In one embodiment, the nucleic acid molecule is incorporated into aviral particle.

In one embodiment, the immunogenic composition further comprises apharmaceutically acceptable excipient.

In one embodiment, the immunogenic composition further comprises anadjuvant.

In one embodiment, the invention relates to a nucleic acid moleculeencoding a peptide comprising an amino acid sequence of at least one ofa) an amino acid sequence having at least about 90% identity over anentire length of the amino acid sequence to at least one of SEQ ID NO:2,SEQ ID NO:4 or SEQ ID NO:6, b) an immunogenic fragment comprising atleast about 90% identity over at least 60% of the amino acid sequence toat least one of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, c) the aminoacid sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, or d) animmunogenic fragment comprising at least 60% of the amino acid sequenceof SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.

In one embodiment, the nucleic acid molecule is a DNA molecule or a RNAmolecule.

In one embodiment, the nucleic acid molecule comprises a nucleotidesequence at least one of a) a nucleotide sequence having at least about90% identity over an entire length of a nucleotide sequence to at leastone of SEQ ID NO: 1, SEQ ID NO:3 or SEQ ID NO:5, b) an immunogenicfragment of a nucleotide sequence having at least about 90% identityover at least 60% of the nucleotide sequence to at least one of SEQ IDNO: 1, SEQ ID NO:3 or SEQ ID NO:5, c) a nucleotide sequence of SEQ IDNO:1, SEQ ID NO:3 or SEQ ID NO:5, or d) an immunogenic fragment of anucleotide sequence of SEQ ID NO: 1, SEQ ID NO:3 or SEQ ID NO:5.

In one embodiment, the nucleotide sequence encoding the peptide isoperably linked to at least one regulatory sequence. In one embodiment,the regulatory sequence is at least one of a start codon, an IgE leadersequence or a stop codon.

In one embodiment, the nucleic acid molecule encodes a peptidecomprising an amino acid sequence of at least one of a) an amino acidsequence having at least about 90% identity over an entire length of theamino acid sequence to at least one of SEQ ID NO:2, SEQ ID NO:4 or SEQID NO:6, b) an immunogenic fragment comprising at least about 90%identity over at least 60% of the amino acid sequence to at least one ofSEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, c) the amino acid sequence ofSEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, or d) an immunogenic fragmentcomprising at least 60% of the amino acid sequence of SEQ ID NO:2, SEQID NO:4 or SEQ ID NO:6, operably linked to an amino acid sequence as setforth in SEQ ID NO:7.

In one embodiment, the nucleic acid molecule comprises a nucleotidesequence of at least one of a) a nucleotide sequence having at leastabout 90% identity over an entire length of a nucleotide sequence to atleast one of SEQ ID NO: 1, SEQ ID NO:3 or SEQ ID NO:5, b) an immunogenicfragment of a nucleotide sequence having at least about 90% identityover at least 60% of the nucleotide sequence to at least one of SEQ IDNO: 1, SEQ ID NO:3 or SEQ ID NO:5, c) a nucleotide sequence of SEQ IDNO:1, SEQ ID NO:3 or SEQ ID NO:5, or d) an immunogenic fragment of anucleotide sequence of SEQ ID NO: 1, SEQ ID NO:3 or SEQ ID NO:5,operably linked to an nucleotide sequence encoding SEQ ID NO:7.

In one embodiment, the nucleic acid molecule comprises an expressionvector.

In one embodiment, the nucleic acid molecule is incorporated into aviral particle.

In one embodiment, the invention relates to an immunogenic compositioncomprising a peptide, wherein the peptide comprises an amino acidsequence of at least one of a) an amino acid sequence having at leastabout 90% identity over an entire length of the amino acid sequence toat least one of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, b) animmunogenic fragment comprising at least about 90% identity over atleast 60% of the amino acid sequence to at least one of SEQ ID NO:2, SEQID NO:4 or SEQ ID NO:6, c) the amino acid sequence of SEQ ID NO:2, SEQID NO:4 or SEQ ID NO:6, or d) an immunogenic fragment comprising atleast 60% of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQID NO:6.

In one embodiment, the invention relates to a peptide, wherein thepeptide comprises an amino acid sequence of at least one of a) an aminoacid sequence having at least about 90% identity over an entire lengthof the amino acid sequence to at least one of SEQ ID NO:2, SEQ ID NO:4or SEQ ID NO:6, b) an immunogenic fragment comprising at least about 90%identity over at least 60% of the amino acid sequence to at least one ofSEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, c) the amino acid sequence ofSEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, or d) an immunogenic fragmentcomprising at least 60% of the amino acid sequence of SEQ ID NO:2, SEQID NO:4 or SEQ ID NO:6.

In one embodiment, the invention relates to a method of inducing animmune response against a MCV T antigen in a subject in need thereof,the method comprising administering an immunogenic compositioncomprising a nucleic acid molecule encoding a modified Merkel CellPolyomavirus (MCV) T antigen, wherein the T antigen comprises at leastone mutation that disrupts at least one oncogenic feature of a nativeMCV T antigen, to the subject.

In one embodiment, the method of administering includes at least one ofelectroporation or injection.

In one embodiment, the invention relates to a method of treating orpreventing a MCV associated pathology in subject in need thereof, themethod comprising administering an immunogenic composition comprising anucleic acid molecule encoding a modified Merkel Cell Polyomavirus (MCV)T antigen, wherein the T antigen comprises at least one mutation thatdisrupts at least one oncogenic feature of a native MCV T antigen, tothe subject.

In one embodiment, the method of administering includes at least one ofelectroporation or injection.

In one embodiment, the MCV associated pathology is at least one of MCVinfection or Merkel Cell Carcinoma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 , comprising FIG. 1A through FIG. 1B, provides schematic diagramsof the LTAg and STAg. FIG. 1A depicts the oncogenic features of the LTAgand STAg. FIG. 1B depicts that the design of the LTAg and STAg of thenucleic acid vaccine incorporate several mutations to disrupt theoncogenic features. *D44N- blocks binding to chaperone proteins; *W209A-blocks binding to hVam6p; *E216K- blocks binding to Rb and preventstransformation; *L142A- blocks binding to PP2A;*91-95LKDYM->AAAAA-blocks binding to Fbxw7 and prevents transformation.

FIG. 2 , comprising FIG. 2A through FIG. 2B, provides schematic diagramsof the consensus LTAg and STAg. FIG. 2A depicts a diagram of theconsensus sequence of the LTAg designed from all available NCBI LTAgsequences. FIG. 2B depicts a diagram of the consensus sequence of theSTAg designed from all available NCBI STAg sequences. These antigensequences were synthesized and cloned into a mammalian expressionplasmid, creating plasmid DNA constructs for expression of syntheticconsensus antigens in vivo.

FIG. 3 depicts exemplary experimental data demonstrating expression ofthe consensus MCC LTAg in vitro. Expression of the consensus MCC STAgwas not detected due to the lack of an effective antibody targeting theSTAg.

FIG. 4 , comprising FIG. 4A through FIG. 4B, provides exemplaryexperimental data demonstrating induction of an immune responsefollowing vaccination with LTAg and STAg alone or in combination. FIG.4A depicts the experimental design. Mice received plasmid DNA followedby intramuscular electroporation at day 0, day 14 and day 28. One weeklater, splenocytes were collected for analysis. Four groups of mice werevaccinated: group 1 - pVax- empty vector control; group 2 - LTAgvaccine; group 3 - STAg vaccine; group 4 - LTAg and STAg vaccine at samesite. FIG. 4B depicts experimental data showing that an induction of animmune response following vaccination with LTAg and STAg alone or incombination, but not following vaccination with an empty control vector(pVax). For these experiments, the peptides were matched to thecorresponding sequences without inactivating mutations.

FIG. 5 , comprising FIG. 5A through FIG. 5B, provides exemplaryexperimental data characterizing the immunodominant epitopes for theLTAg and STAg. FIG. 5A depicts the immunodominant epitopes for LTAgvaccination. FIG. 5B depicts the immunodominant epitopes for STAgvaccination.

FIG. 6 depicts the results on an analysis of the extent of MCC Large Ttruncation in human Merkel cell carcinoma samples. Data was compiledfrom 42 Large T sequences in GenBank.

FIG. 7 , comprising FIG. 7A through FIG. 7F, provides exemplaryexperimental data demonstrating the levels of CD4⁺ and CD8⁺ T cellresponses for cytokines following vaccination and stimulation for 5hours with LTAg peptides. FIG. 7A depicts the levels of CD8⁺ T cellresponse for IFNy. FIG. 7B depicts the levels of CD8⁺ T cell responsefor TNFα. FIG. 7C depicts the levels of CD8⁺ T cell response for IL-2.FIG. 7D depicts the levels of CD4⁺ T cell response for IFNy. FIG. 7Edepicts the levels of CD4⁺ T cell response for TNFα. FIG. 7F depicts thelevels of CD4⁺ T cell response for IL-2.

FIG. 8 depicts exemplary experimental data demonstrating that LTAgvaccination induces robust polyfunctional CD8 T cells.

FIG. 9 depicts exemplary experimental data demonstrating that LTAgvaccination induces robust polyfunctional CD4 T cells.

FIG. 10 depicts exemplary experimental data demonstrating that LTAgvaccination induces CD8 T cells with cytotoxic potential that co-expressCD107a, IFNγ and T-bet.

FIG. 11 depicts exemplary experimental data demonstrating that Large Tand Small T antigen vaccines generate humoral responses, demonstratedusing mouse serum as a primary antibody.

FIG. 12 depicts exemplary experimental data demonstrating that the LTAgvaccine induces robust immune responses in genetically diverse, CD-1outbred mice.

FIG. 13 depicts exemplary experimental data demonstrating that the STAgvaccine induces immune responses in genetically diverse, CD-1 outbredmice.

FIG. 14 , comprising FIG. 14A through FIG. 14F, provides exemplaryexperimental data demonstrating the levels of CD4⁺ and CD8⁺ T cellresponses for cytokines following vaccination in CD-1 outbred mice andstimulation for 5 hours with LTAg peptides. FIG. 14A depicts the levelsof CD8⁺ T cell response for IFNy. FIG. 14B depicts the levels of CD8⁺ Tcell response for TNFα. FIG. 14C depicts the levels of CD8⁺ T cellresponse for IL-2. FIG. 14D depicts the levels of CD4⁺ T cell responsefor IFNy. FIG. 14E depicts the levels of CD4⁺ T cell response for TNFα.FIG. 14F depicts the levels of CD4⁺ T cell response for IL-2.

FIG. 15 , comprising FIG. 15A through FIG. 15F, provides exemplaryexperimental data demonstrating the levels of CD4⁺ and CD8⁺ T cellresponses for cytokines following vaccination in CD-1 outbred mice andstimulation for 5 hours with STAg peptides. FIG. 15A depicts the levelsof CD8⁺ T cell response for IFNy. FIG. 15B depicts the levels of CD8⁺ Tcell response for TNFα. FIG. 15C depicts the levels of CD8⁺ T cellresponse for IL-2. FIG. 15D depicts the levels of CD4⁺ T cell responsefor IFNy. FIG. 15E depicts the levels of CD4⁺ T cell response for TNFα.FIG. 15F depicts the levels of CD4⁺ T cell response for IL-2.

DETAILED DESCRIPTION

Merkel Cell Polyomavirus (MCV) infection is associated with Merkel CellCarcinoma (MCC), which currently has a 46% mortality rate.

In one embodiment, the invention includes a nucleic acid vaccine againstMCV and MCC. In one embodiment, the vaccine comprise a plasmid encodinga consensus MCV T antigen. In one embodiment, the consensus MCV Tantigen is a large T antigen (LTAg). In one embodiment, the consensusMCV T antigen is a small t antigen (STAg). In one embodiment, theconsensus MCV T antigens further comprise mutations that disrupt theoncogenic features of native T antigens. As a vaccine candidate, anenhanced DNA (DNA)-based platform provides many advantages in geneticoptimization and delivery techniques. As such, each MCV T antigen can begenetically-optimized, subcloned into modified mammalian expressionvectors, and then delivered using in vivo electroporation (EP).

Vaccination in preclinical rodent studies was highly potent, asvaccination with synthetic consensus MCV T antigen constructs generatesrobust immune responses.

In some embodiments, the strategy employs a coding sequence for asynthetic consensus MCV T antigen. Coding sequence for a LTAg and a STAgare provided. In some embodiments, the strategy employs coding sequencesfor a single synthetic consensus MCV T antigen. In some embodiments, thestrategy employs coding sequences for multiple synthetic consensus MCV Tantigens.

As a candidate for vaccines, DNA vaccines exhibit a multitude ofadvantages including rapid and inexpensive up-scale production,stability at room temperature, and ease of transport, all of whichfurther enhance this platform from an economic and geographicperspective. Due to the synthetic nature of the plasmids, antigensequences can be quickly and easily modified in response to newlyemergent strains and/or expanded to include additional vaccinecomponents.

Optimization of plasmid DNA vectors and their encoded antigen genes haveled to increases in in vivo immunogenicity. Cellular uptake andsubsequent antigen expression are substantially amplified whenhighly-concentrated plasmid vaccine formulations are administered within vivo electroporation, a technology that uses brief square-waveelectric pulses within the vaccination site to drive plasmids intotransiently permeabilized cells. In theory, a cocktail of DNA plasmidscould be assembled for directing a highly-specialized immune responseagainst any number of variable antigens. Immunity can be furtherdirected by co-delivery with plasmid molecular adjuvants encodingspecies-specific cytokine genes as well as ‘consensus-engineering’ ofthe antigen amino acid sequences to help bias vaccine-induced immunitytowards particular strains.

1. Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting.

The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The singular forms“a,” “and” and “the” include plural references unless the contextclearly dictates otherwise. The present disclosure also contemplatesother embodiments “comprising,” “consisting of” and “consistingessentially of,” the embodiments or elements presented herein, whetherexplicitly set forth or not.

“Adjuvant” as used herein may mean any molecule added to a nucleic acidvaccines to enhance antigenicity of the vaccine.

“Antibody” may mean an antibody of classes IgG, IgM, IgA, IgD or IgE, orfragments, fragments or derivatives thereof, including Fab, F(ab′)2, Fd,and single chain antibodies, diabodies, bispecific antibodies,bifunctional antibodies and derivatives thereof. The antibody may be anantibody isolated from the serum sample of mammal, a polyclonalantibody, affinity purified antibody, or mixtures thereof which exhibitssufficient binding specificity to a desired epitope or a sequencederived therefrom.

“Antibody fragment” or “fragment of an antibody” as used interchangeablyherein refers to a portion of an intact antibody comprising theantigen-binding site or variable region. The portion does not includethe constant heavy chain domains (i.e. CH2, CH3, or CH4, depending onthe antibody isotype) of the Fc region of the intact antibody. Examplesof antibody fragments include, but are not limited to, Fab fragments,Fab′ fragments, Fab′-SH fragments, F(ab′)2 fragments, Fd fragments, Fvfragments, diabodies, single-chain Fv (scFv) molecules, single-chainpolypeptides containing only one light chain variable domain,single-chain polypeptides containing the three CDRs of the light-chainvariable domain, single-chain polypeptides containing only one heavychain variable region, and single-chain polypeptides containing thethree CDRs of the heavy chain variable region.

“Antigen” refers to proteins that have the ability to generate an immuneresponse in a host. An antigen may be recognized and bound by anantibody. An antigen may originate from within the body or from theexternal environment.

“Coding sequence” or “encoding nucleic acid” as used herein may meanrefers to the nucleic acid (RNA or DNA molecule) that comprise anucleotide sequence which encodes a protein. The coding sequence mayfurther include initiation and termination signals operably linked toregulatory elements including a promoter and polyadenylation signalcapable of directing expression in the cells of an individual or mammalto whom the nucleic acid is administered. The coding sequence mayoptionally further comprise a start codon that encodes an N terminalmethionine or a signal peptide such as an IgE or IgG signal peptide.

“Complement” or “complementary” as used herein may mean a nucleic acidmay mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairingbetween nucleotides or nucleotide analogs of nucleic acid molecules.

“Consensus” or “consensus sequence” as used herein may mean a syntheticnucleotide sequence, or corresponding polypeptide sequence, constructedbased on analysis of an alignment of multiple sequences (e.g., multiplesequences of a particular virus antigen.)

“Constant current” as used herein to define a current that is receivedor experienced by a tissue, or cells defining said tissue, over theduration of an electrical pulse delivered to same tissue. The electricalpulse is delivered from the electroporation devices described herein.This current remains at a constant amperage in said tissue over the lifeof an electrical pulse because the electroporation device providedherein has a feedback element, preferably having instantaneous feedback.The feedback element can measure the resistance of the tissue (or cells)throughout the duration of the pulse and cause the electroporationdevice to alter its electrical energy output (e.g., increase voltage) socurrent in same tissue remains constant throughout the electrical pulse(on the order of microseconds), and from pulse to pulse. In someembodiments, the feedback element comprises a controller.

“Current feedback” or “feedback” as used herein may be usedinterchangeably and may mean the active response of the providedelectroporation devices, which comprises measuring the current in tissuebetween electrodes and altering the energy output delivered by the EPdevice accordingly in order to maintain the current at a constant level.This constant level is preset by a user prior to initiation of a pulsesequence or electrical treatment. The feedback may be accomplished bythe electroporation component, e.g., controller, of the electroporationdevice, as the electrical circuit therein is able to continuouslymonitor the current in tissue between electrodes and compare thatmonitored current (or current within tissue) to a preset current andcontinuously make energy-output adjustments to maintain the monitoredcurrent at preset levels. The feedback loop may be instantaneous as itis an analog closed-loop feedback.

“Decentralized current” as used herein may mean the pattern ofelectrical currents delivered from the various needle electrode arraysof the electroporation devices described herein, wherein the patternsminimize, or preferably eliminate, the occurrence of electroporationrelated heat stress on any area of tissue being electroporated.

“Electroporation,” “electro-permeabilization,” or “electro-kineticenhancement” (“EP”) as used interchangeably herein may refer to the useof a transmembrane electric field pulse to induce microscopic pathways(pores) in a bio-membrane; their presence allows biomolecules such asplasmids, oligonucleotides, siRNA, drugs, ions, and water to pass fromone side of the cellular membrane to the other.

“Endogenous antibody” as used herein may refer to an antibody that isgenerated in a subject that is administered an effective dose of anantigen for induction of a humoral immune response.

“Feedback mechanism” as used herein may refer to a process performed byeither software or hardware (or firmware), which process receives andcompares the impedance of the desired tissue (before, during, and/orafter the delivery of pulse of energy) with a present value, preferablycurrent, and adjusts the pulse of energy delivered to achieve the presetvalue. A feedback mechanism may be performed by an analog closed loopcircuit.

“Fragment” may mean a percentage of a full length polypeptide sequenceor nucleotide sequence. Fragments may comprise 20% or more, 25% or more,30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% ormore, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more,85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% ormore, 95% or more, 96% or more, 97% or more, 98% or more, 99% or morepercent of the full length of the parental nucleotide sequence or aminoacid sequence or variant thereof.

“Genetic construct” as used herein refers to the DNA or RNA moleculesthat comprise a nucleotide sequence which encodes a protein, such as anantibody. The genetic construct may also refer to a DNA molecule whichtranscribes an RNA. The coding sequence includes initiation andtermination signals operably linked to regulatory elements including apromoter and polyadenylation signal capable of directing expression inthe cells of the individual to whom the nucleic acid molecule isadministered. As used herein, the term “expressible form” refers to geneconstructs that contain the necessary regulatory elements operablelinked to a coding sequence that encodes a protein such that whenpresent in the cell of the individual, the coding sequence will beexpressed.

“Identical” or “identity” as used herein in the context of two or morenucleic acids or polypeptide sequences, may mean that the sequences havea specified percentage of residues that are the same over a specifiedregion. The percentage may be calculated by optimally aligning the twosequences, comparing the two sequences over the specified region,determining the number of positions at which the identical residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the specified region, and multiplying the result by 100 toyield the percentage of sequence identity. In cases where the twosequences are of different lengths or the alignment produces one or morestaggered ends and the specified region of comparison includes only asingle sequence, the residues of single sequence are included in thedenominator but not the numerator of the calculation. When comparing DNAand RNA, thymine (T) and uracil (U) may be considered equivalent.Identity may be performed manually or by using a computer sequencealgorithm such as BLAST or BLAST 2.0.

“Impedance” as used herein may be used when discussing the feedbackmechanism and can be converted to a current value according to Ohm’slaw, thus enabling comparisons with the preset current.

“Immune response” as used herein may mean the activation of a host’simmune system, e.g., that of a mammal, in response to the introductionof one or more consensus antigen via the provided vaccines. The immuneresponse can be in the form of a cellular or humoral response, or both.

“Nucleic acid” or “oligonucleotide” or “polynucleotide” as used hereinmay mean at least two nucleotides covalently linked together. Thedepiction of a single strand also defines the sequence of thecomplementary strand. Thus, a nucleic acid also encompasses thecomplementary strand of a depicted single strand. Many variants of anucleic acid may be used for the same purpose as a given nucleic acid.Thus, a nucleic acid also encompasses substantially identical nucleicacids and complements thereof. A single strand provides a probe that mayhybridize to a target sequence under stringent hybridization conditions.Thus, a nucleic acid also encompasses a probe that hybridizes understringent hybridization conditions.

Nucleic acids may be single stranded or double stranded, or may containportions of both double stranded and single stranded sequence. Thenucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, wherethe nucleic acid may contain combinations of deoxyribo- andribo-nucleotides, and combinations of bases including uracil, adenine,thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosineand isoguanine. Nucleic acids may be obtained by chemical synthesismethods or by recombinant methods.

“Operably linked” as used herein may mean that expression of a gene isunder the control of a promoter with which it is spatially connected. Apromoter may be positioned 5′ (upstream) or 3′ (downstream) of a geneunder its control. The distance between the promoter and a gene may beapproximately the same as the distance between that promoter and thegene it controls in the gene from which the promoter is derived. As isknown in the art, variation in this distance may be accommodated withoutloss of promoter function.

A “peptide,” “protein,” or “polypeptide” as used herein can mean alinked sequence of amino acids and can be natural, synthetic, or amodification or combination of natural and synthetic.

“Promoter” as used herein may mean a synthetic or naturally-derivedmolecule which is capable of conferring, activating or enhancingexpression of a nucleic acid in a cell. A promoter may comprise one ormore specific transcriptional regulatory sequences to further enhanceexpression and/or to alter the spatial expression and/or temporalexpression of same. A promoter may also comprise distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A promoter may bederived from sources including viral, bacterial, fungal, plants,insects, and animals. A promoter may regulate the expression of a genecomponent constitutively, or differentially with respect to cell, thetissue or organ in which expression occurs or, with respect to thedevelopmental stage at which expression occurs, or in response toexternal stimuli such as physiological stresses, pathogens, metal ions,or inducing agents. Representative examples of promoters include thebacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lacoperator-promoter, tac promoter, SV40 late promoter, SV40 earlypromoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40late promoter and the CMV IE promoter.

“Signal peptide” and “leader sequence” are used interchangeably hereinand refer to an amino acid sequence that can be linked at the aminoterminus of a protein set forth herein. Signal peptides/leader sequencestypically direct localization of a protein. Signal peptides/leadersequences used herein preferably facilitate secretion of the proteinfrom the cell in which it is produced. Signal peptides/leader sequencesare often cleaved from the remainder of the protein, often referred toas the mature protein, upon secretion from the cell. Signalpeptides/leader sequences are linked at the N terminus of the protein.

“Stringent hybridization conditions” as used herein may mean conditionsunder which a first nucleic acid molecule (e.g., probe) will hybridizeto a second nucleic acid molecule (e.g., target), such as in a complexmixture of nucleic acids. Stringent conditions are sequence-dependentand will be different in different circumstances. Stringent conditionsmay be selected to be about 5-10° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence at a defined ionic strength pH.The T_(m) may be the temperature (under defined ionic strength, pH, andnucleic concentration) at which 50% of the probes complementary to thetarget hybridize to the target sequence at equilibrium (as the targetsequences are present in excess, at T_(m), 50% of the probes areoccupied at equilibrium). Stringent conditions may be those in which thesalt concentration is less than about 1.0 M sodium ion, such as about0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3and the temperature is at least about 30° C. for short probes (e.g.,about 10-50 nucleotides) and at least about 60° C. for long probes(e.g., greater than about 50 nucleotides). Stringent conditions may alsobe achieved with the addition of destabilizing agents such as formamide.For selective or specific hybridization, a positive signal may be atleast 2 to 10 times background hybridization. Exemplary stringenthybridization conditions include the following: 50% formamide, 5x SSC,and 1% SDS, incubating at 42° C., or, 5x SSC, 1% SDS, incubating at 65°C., with wash in 0.2x SSC, and 0.1% SDS at 65° C.

“Subject” and “patient” as used herein interchangeably refers to anyvertebrate, including, but not limited to, a mammal (e.g., cow, pig,camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat,dog, rat, and mouse, a non-human primate (for example, a monkey, such asa cynomolgous or rhesus monkey, chimpanzee, etc) and a human). In someembodiments, the subject may be a human or a non-human.

“Substantially complementary” as used herein may mean that a firstsequence is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the complement of a second sequence over a region of 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotidesor amino acids, or that the two sequences hybridize under stringenthybridization conditions.

“Substantially identical” as used herein may mean that a first andsecond sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%,or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 1100 or more nucleotides or amino acids, or with respect tonucleic acids, if the first sequence is substantially complementary tothe complement of the second sequence.

“Treatment” or “treating,” as used herein can mean protecting of asubject from a disease through means of preventing, suppressing,repressing, or completely eliminating the disease. Preventing thedisease involves administering a vaccine of the present invention to asubject prior to onset of the disease. Suppressing the disease involvesadministering a vaccine of the present invention to a subject afterinduction of the disease but before its clinical appearance. Repressingthe disease involves administering a vaccine of the present invention toa subject after clinical appearance of the disease.

“Variant” as used herein with respect to a nucleic acid may mean (i) aportion or fragment of a referenced nucleotide sequence; (ii) thecomplement of a referenced nucleotide sequence or portion thereof; (iii)a nucleic acid that is substantially identical to a referenced nucleicacid or the complement thereof; or (iv) a nucleic acid that hybridizesunder stringent conditions to the referenced nucleic acid, complementthereof, or a sequences substantially identical thereto.

“Variant” with respect to a peptide or polypeptide that differs in aminoacid sequence by the insertion, deletion, or conservative substitutionof amino acids, but retain at least one biological activity. Variant mayalso mean a protein with an amino acid sequence that is substantiallyidentical to a referenced protein with an amino acid sequence thatretains at least one biological activity. A conservative substitution ofan amino acid, i.e., replacing an amino acid with a different amino acidof similar properties (e.g., hydrophilicity, degree and distribution ofcharged regions) is recognized in the art as typically involving a minorchange. These minor changes can be identified, in part, by consideringthe hydropathic index of amino acids, as understood in the art. Kyte etal., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an aminoacid is based on a consideration of its hydrophobicity and charge. It isknown in the art that amino acids of similar hydropathic indexes can besubstituted and still retain protein function. In one aspect, aminoacids having hydropathic indexes of ±2 are substituted. Thehydrophilicity of amino acids can also be used to reveal substitutionsthat would result in proteins retaining biological function. Aconsideration of the hydrophilicity of amino acids in the context of apeptide permits calculation of the greatest local average hydrophilicityof that peptide, a useful measure that has been reported to correlatewell with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101,incorporated fully herein by reference. Substitution of amino acidshaving similar hydrophilicity values can result in peptides retainingbiological activity, for example immunogenicity, as is understood in theart. Substitutions may be performed with amino acids havinghydrophilicity values within ±2 of each other. Both the hyrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties.

A variant may be a nucleotide sequence that is substantially identicalover the full length of the full gene sequence or a fragment thereof.The nucleotide sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical over the full length of the gene sequence or a fragmentthereof. A variant may be an amino acid sequence that is substantiallyidentical over the full length of the amino acid sequence or fragmentthereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical over the full length of the amino acid sequence or afragment thereof.

“Vector” as used herein may mean a nucleic acid molecule containing anorigin of replication. A vector may be a plasmid, bacteriophage,bacterial artificial chromosome or yeast artificial chromosome. A vectormay be a DNA or RNA vector. A vector may be either a self-replicatingextrachromosomal vector or a vector which integrates into a host genome.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

2. Description

The invention provides an optimized consensus sequence encoding a MCV Tantigen. In one embodiment, the MCV T antigen encoded by the optimizedconsensus sequence is capable of eliciting an immune response in amammal. In one embodiment, the MCV T antigen encoded by the optimizedconsensus sequence can comprise an epitope(s) that makes it particularlyeffective as an immunogen against which an immune response can beinduced.

The optimized consensus sequence can be a consensus sequence derivedfrom two or more MCV T antigens. The optimized consensus sequence cancomprise a consensus sequence and/or modification(s) for improvedexpression. Modification can include codon optimization, RNAoptimization, addition of a kozak sequence for increased translationinitiation, and/or the addition of an immunoglobulin leader sequence toincrease immunogenicity. The MCV T antigen encoded by the optimizedconsensus sequence can comprise a signal peptide such as animmunoglobulin signal peptide, for example, but not limited to, animmunoglobulin E (IgE) or immunoglobulin (IgG) signal peptide. In someembodiments, the antigen encoded by the optimized consensus sequence cancomprise a hemagglutinin (HA) tag. The antigen encoded by the optimizedconsensus sequence can be designed to elicit stronger cellular and/orhumoral immune responses than a corresponding non-optimized antigen.

Provided herein are MCV T antigens that can be used to induce immunityagainst MCV in genetically diverse subjects with MCV infection. In oneembodiment, the present invention provides an immunogenic compositioncomprising one or more nucleic acid molecules that are capable ofgenerating in a mammal an immune response against a MCV T antigen. Thepresent invention also provides isolated nucleic acid molecules that arecapable of generating in a mammal an immune response against a MCV Tantigen. In one embodiment, the nucleic acid molecule comprises anoptimized nucleotide sequence encoding a consensus MCV T antigen.

In one embodiment, the MCV T antigens are modified to reduce or disruptat least one oncogenic feature of a native MCV T antigen. In variousembodiments, the MCV T antigens are modified to reduce or disrupt atleast one of CR1 binding, DnaJ binding, phophatase pp2A-binding binding,Rb binding, ATPase activity, helicase activity, chaperone proteinbinding, hVam6p binding, Fbxw7 binding, origin binding, andtransformation. In one embodiment, the MCV T antigen comprises at leastone mutation at D44, W209, E216, L142, L91, K92, D93, Y94 or M95relative to the native T antigen sequence. In one embodiment, the MCV Tantigen comprises at least one of a D44N mutation, a W209A, an E216Kmutation, an L142A mutation, an L91A mutation, a K92A mutation, a D93Amutation, a Y94A mutation and a M95A mutation. In one embodiment, theMCV LTAg comprises at least one of a D44N mutation, a W209A, and anE216K mutation. In one embodiment, the MCV LTAg comprises a D44Nmutation, a W209A, and an E216K mutation. In one embodiment, the MCVSTAg comprises at least one of a D44N mutation, an L142A mutation, anL91A mutation, a K92A mutation, a D93A mutation, a Y94A mutation and aM95A mutation. In one embodiment, the MCV STAg comprises a D44Nmutation, an L142A mutation, an L91A mutation, a K92A mutation, a D93Amutation, a Y94A mutation and a M95A mutation.

Consensus amino acid sequences for MCV T antigens include SEQ ID NO:2,SEQ ID NO:4, and variants thereof and fragments of SEQ ID NO:2, SEQ IDNO:4, and variants thereof. An exemplary amino acid sequence of amodified synthetic consensus MCV LTAg is provided as SEQ ID NO:2. Anexemplary amino acid sequence of a modified synthetic consensus MCV STAgis provided as SEQ ID NO:2.

In one embodiment, the invention provides compositions comprising anucleic acid molecule comprising a nucleotide sequence that encodes amodified synthetic consensus MCV T antigen. In one embodiment, anucleotide sequence which encodes a modified synthetic consensus MCVLTAg is provided as SEQ ID NO:1, which encodes SEQ ID NO:2. In oneembodiment, a nucleotide sequence which encodes a modified syntheticconsensus MCV STAg is provided as SEQ ID NO:3, which encodes SEQ IDNO:4.

In various embodiments, the invention provides compositions comprising acombination of a modified LTAg and a modified STAg, or one or morenucleic acid molecules encoding the same. The compositions may comprisea plurality of copies of a single nucleic acid molecule such a singleplasmid, or a plurality of copies of two or more different nucleic acidmolecules such as two or more different plasmids.

Compositions may comprise a single nucleic acid molecule, such as aplasmid, that contains coding sequence for multiple consensus MCV Tantigens. In one embodiment, the compositions may comprise a singlenucleic acid molecule comprising nucleotide sequences that encode a MCVLTAg and a MCV STAg. In one embodiment, each coding sequence for eachconsensus MCV T antigen is on a separate plasmid.

Accordingly, compositions that comprise one or more nucleotide sequencethat encode multiple consensus MCV T antigens may be on a singleplasmid. In one embodiment, a composition comprises a single plasmidthat encodes a MCV LTAg and a MCV STAg under a single promoter. In suchan embodiment, the sequence that encodes the MCV LTAg and the sequencethat encodes the MCV STAg may be linked by a fusion peptide sequence,for example a furin cleavage sequence. An exemplary amino acid sequenceof a single construct comprising a modified synthetic consensus MCV LTAgand MCV STAg linked by a furin cleavage site is provided as SEQ ID NO:6.In one embodiment, a single nucleotide sequence which encodes a modifiedsynthetic consensus MCV LTAg and MCV STAg linked by a furin cleavagesequence is provided as SEQ ID NO:5, which encodes SEQ ID NO:6.

In one embodiment, an optimized consensus encoded MCV T antigen isoperably linked to one or more regulatory elements. In one embodiment, aregulatory element is a leader sequence. In one embodiment, the leadersequence is an IgE leader sequence. In one embodiment, the IgE leadersequence has an amino acid sequence as set forth in SEQ ID NO:7.Therefore in one embodiment, the invention relates to an amino acidsequence as set forth in SEQ ID NO:2, SEQ ID NO: 4 or SEQ ID NO:6operably linked to an amino acid sequence as set forth in SEQ ID NO:7.In one embodiment, the invention relates to a nucleotide sequenceencoding an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 operably linked to an amino acid sequence as set forthin SEQ ID NO:7.

In one embodiment, a regulatory element is a start codon. Therefore, inone embodiment, the invention relates to a nucleotide sequence as setforth in SEQ ID NO: 1, SEQ ID NO:3 or SEQ ID NO:5, or a fragment orhomolog thereof, operably linked to a nucleotide sequence comprising astart codon at the 5′ terminus. In one embodiment, the invention relatesto an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4 orSEQ ID NO:6 or a fragment or homolog thereof, operably linked to anamino acid encoded by a start codon (e.g., a Methionine) at theN-terminus.

In one embodiment, a regulatory element is at least one stop codon.Therefore, in one embodiment, the invention relates to a nucleotidesequence as set forth in SEQ ID NO: 1, SEQ ID NO:3 or SEQ ID NO:5, or afragment or homolog thereof, operably linked to a nucleotide sequencecomprising at least one stop codon at the 3′ terminus. In oneembodiment, the nucleotide sequence is operably linked to two stopcodons to increase the efficiency of translational termination.

In one embodiment, nucleic acid molecule can encode a peptide having theamino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4 or SEQ IDNO:6. In one embodiment, the nucleic acid molecule comprises thenucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:3 or SEQ IDNO:5. In some embodiments, the sequence can be the nucleotide sequencehaving at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity overan entire length of the nucleotide sequence set forth in SEQ ID NO: 1,SEQ ID NO:3 or SEQ ID NO:5. In other embodiments, sequence can be thenucleotide sequence that encodes the amino acid sequence having at leastabout 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entirelength of the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4or SEQ ID NO:6.

In some embodiments, the nucleic acid molecule comprises an RNA sequencethat is a transcript from a DNA sequence having at least about 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity over an entire length of thenucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO:3 or SEQ IDNO:5. In some embodiments, the nucleic acid molecule comprises an RNAsequence that encodes an amino acid sequence having at least about 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identity over an entire length of theamino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4 or SEQ IDNO:6.

In some embodiments, the nucleic acid molecule may comprise a nucleotidesequence that encodes a full length consensus MCV T antigen. The nucleicacid molecules may comprise a sequence that encodes SEQ ID NO:2, SEQ IDNO:4 or SEQ ID NO:6. The nucleic acid molecules may comprise anucleotide sequence of SEQ ID NO: 1, SEQ ID NO:3 or SEQ ID NO:5. Thenucleic acid moleclue may optionally comprise coding sequences thatencode a signal peptide such as for example an IgE or IgG signalpeptide.

The consensus-MCV T antigen can be a peptide having the amino acidsequence set forth in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. In someembodiments, the antigen can have an amino acid sequence having at leastabout 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entirelength of the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4or SEQ ID NO:6.

Immunogenic fragments of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 can beprovided. Immunogenic fragments can comprise at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99% ofthe full length of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. In someembodiments, immunogenic fragments include a leader sequence, such asfor example an immunoglobulin leader, such as the IgE leader. In someembodiments, immunogenic fragments are free of a leader sequence.

Immunogenic fragments of proteins with amino acid sequences homologousto immunogenic fragments of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, canbe provided. Such immunogenic fragments can comprise at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% of proteins that are 95% homologous to SEQ ID NO:2, SEQ IDNO:4 or SEQ ID NO:6. Some embodiments relate to immunogenic fragmentsthat have 96% homology to the immunogenic fragments of consensus proteinsequences herein. Some embodiments relate to immunogenic fragments thathave 97% homology to the immunogenic fragments of consensus proteinsequences herein. Some embodiments relate to immunogenic fragments thathave 98% homology to the immunogenic fragments of consensus proteinsequences herein. Some embodiments relate to immunogenic fragments thathave 99% homology to the immunogenic fragments of consensus proteinsequences herein. In some embodiments, immunogenic fragments include aleader sequence, such as for example an immunoglobulin leader, such asthe IgE leader. In some embodiments, immunogenic fragments are free of aleader sequence.

In one embodiment, an immunogenic fragment of a nucleic acid moleculeencodes at least one immunodominant or sub-immunodominant epitope of afull length optimized consensus MCV T antigen.

Some embodiments relate to immunogenic fragments of SEQ ID NO:1, SEQ IDNO:3 or SEQ ID NO:5 comprising at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% of the full lengthof SEQ ID NO: 1, SEQ ID NO:3 or SEQ ID NO:5. Immunogenic fragments canbe at least 96%, at least 97% at least 98% or at least 99% homologous tofragments of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5. In someembodiments, immunogenic fragments include sequences that encode aleader sequence, such as for example an immunoglobulin leader, such asthe IgE leader. In some embodiments, fragments are free of codingsequences that encode a leader sequence.

In one embodiment, the nucleic acid molecule comprises a sequence atleast 90% homologous to SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.

In one embodiment, the nucleic acid molecule comprises an RNA sequenceencoding a consensus MCV T antigen sequence described herein. Forexample, nucleic acids may comprise an RNA sequence encoding one or moreof SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO: 6, a variant thereof, afragment thereof or any combination thereof.

In some embodiments, the nucleic acid molecule includes a sequence thatencodes for a MCV T antigen minus an IgE leader sequence on theN-terminal end of the coding sequence. In some embodiments, the DNAnucleic acid molecule further comprises an IgE leader sequence attachedto an N-terminal end of the coding sequence and operably linked to thepromoter.

The nucleic acid molecule can further include a polyadenylation sequenceattached to the C-terminal end of the coding sequence. In oneembodiment, the nucleic acid molecule is codon optimized.

Vaccines and Immunogenic Compositions

Immunogenic compositions, such as vaccines, are provided comprising anoptimized consensus sequence, an optimized consensus-encoded antigen, afragment thereof, a variant thereof, or a combination thereof. Theimmunogenic composition can significantly induce an immune response of asubject administered with the immunogenic composition against the MCV Tantigen. The vaccine may comprise a plurality of the nucleic acidmolecules, or combinations thereof. The vaccine may be provided toinduce a therapeutic or prophylactic immune response.

The immunogenic composition can be a DNA vaccine, an RNA vaccine, apeptide vaccine, or a combination vaccine. The vaccine can include anoptimized consensus nucleotide sequence encoding an antigen. Thenucleotide sequence can be DNA, RNA, cDNA, a variant thereof, a fragmentthereof, or a combination thereof. The nucleotide sequence can alsoinclude additional sequences that encode linker, leader, or tagsequences that are linked to the antigen by a peptide bond. The peptidevaccine can include an antigen, a variant thereof, a fragment thereof,or a combination thereof. The combination DNA and peptide vaccine caninclude the above described optimized consensus nucleotide sequence andthe encoded antigen.

The vaccine can be a DNA vaccine. DNA vaccines are disclosed in U.S.Pat. Nos. 5,593,972, 5,739,118, 5,817,637, 5,830,876, 5,962,428,5,981,505, 5,580,859, 5,703,055, and 5,676,594, which are incorporatedherein fully by reference. The DNA vaccine can further comprise elementsor reagents that inhibit it from integrating into the chromosome.

The vaccine can be an RNA of the one or more MCV T antigens. The RNAvaccine can be introduced into the cell.

The vaccine can be an attenuated live vaccine, a vaccine usingrecombinant vectors to deliver antigen, subunit vaccines, andglycoprotein vaccines, for example, but not limited, the vaccinesdescribed in U.S. Pat. Nos.: 4,510,245; 4,797,368; 4,722,848; 4,790,987;4,920,209; 5,017,487; 5,077,044; 5,110,587; 5,112,749; 5,174,993;5,223,424; 5,225,336; 5,240,703; 5,242,829; 5,294,441; 5,294,548;5,310,668; 5,387,744; 5,389,368; 5,424,065; 5,451,499; 5,453,364;5,462,734; 5,470,734; 5,474,935; 5,482,713; 5,591,439; 5,643,579;5,650,309; 5,698,202; 5,955,088; 6,034,298; 6,042,836; 6,156,319 and6,589,529, which are each incorporated herein by reference.

The vaccine of the present invention can have features required ofeffective vaccines such as being safe so that the vaccine itself doesnot cause illness or death; being protective against illness; inducingprotective T cell responses; and providing ease of administration, fewside effects, biological stability, and low cost per dose.

Provided herein is an immunogenic composition capable of generating in amammal an immune response against MCV. The immunogenic composition maycomprise each plasmid as discussed above. The immunogenic compositionmay comprise a plurality of the plasmids, or combinations thereof. Theimmunogenic composition may be provided to induce a therapeutic orprophylactic immune response.

Immunogenic compositions may be used to deliver nucleic acid moleculesthat encode one or more consensus MCV T antigen. Immunogeniccompositions are preferably compositions comprising plasmids.

The immunogenic composition may further comprise a pharmaceuticallyacceptable excipient. The pharmaceutically acceptable excipient may befunctional molecules as vehicles, adjuvants, carriers, or diluents. Thepharmaceutically acceptable excipient may be a transfection facilitatingagent, which may include surface active agents, such asimmune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPSanalog including monophosphoryl lipid A, muramyl peptides, quinoneanalogs, vesicles such as squalene and squalene, hyaluronic acid,lipids, liposomes, calcium ions, viral proteins, polyanions,polycations, or nanoparticles, or other known transfection facilitatingagents.

The transfection facilitating agent is a polyanion, polycation,including poly-L-glutamate (LGS), or lipid. The transfectionfacilitating agent is poly-L-glutamate, and more preferably, thepoly-L-glutamate is present in the immunogenic composition at aconcentration less than 6 mg/ml. The transfection facilitating agent mayalso include surface active agents such as immune-stimulating complexes(ISCOMS), Freunds incomplete adjuvant, LPS analog includingmonophosphoryl lipid A, muramyl peptides, quinone analogs and vesiclessuch as squalene and squalene, and hyaluronic acid may also be usedadministered in conjunction with the genetic construct. In someembodiments, the immunogenic compositions may also include atransfection facilitating agent such as lipids, liposomes, includinglecithin liposomes or other liposomes known in the art, as aDNA-liposome mixture (see for example W09324640), calcium ions, viralproteins, polyanions, polycations, or nanoparticles, or other knowntransfection facilitating agents. Preferably, the transfectionfacilitating agent is a polyanion, polycation, includingpoly-L-glutamate (LGS), or lipid. Concentration of the transfectionagent in the immunogenic composition is less than 4 mg/ml, less than 2mg/ml, less than 1 mg/ml, less than 0.750 mg/ml, less than 0.500 mg/ml,less than 0.250 mg/ml, less than 0.100 mg/ml, less than 0.050 mg/ml, orless than 0.010 mg/ml.

The pharmaceutically acceptable excipient may be one or more adjuvants.An adjuvant may be other genes that are expressed from the same or froman alternative plasmid or are delivered as proteins in combination withthe plasmid above in the immunogenic composition. The one or moreadjuvants may be proteins and/or nucleic acid molecules that encodeproteins selected from the group consisting of: CCL20, α-interferon(IFN- α), β-interferon (IFN-β), γ-interferon, platelet derived growthfactor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF),cutaneous T cell-attracting chemokine (CTACK), epithelialthymus-expressed chemokine (TECK), mucosae-associated epithelialchemokine (MEC), IL-12, IL-15 including IL-15 having the signal sequenceor coding sequence that encodes the signal sequence deleted andoptionally including a different signal peptide such as that from IgE orcoding sequence that encodes a difference signal peptide such as thatfrom IgE, IL-28, MHC, CD80, CD86, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10,IL-18, MCP-1, MIP-lα, MIP-1β, IL-8, L-selectin, P-selectin, E-selectin,CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1,ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, mutant forms of IL-18, CD40,CD40L, vascular growth factor, fibroblast growth factor, IL-7, nervegrowth factor, vascular endothelial growth factor, Fas, TNF receptor,Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5,KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1,Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1,JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec,TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND,NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 andfunctional fragments thereof. or a combination thereof.

In some embodiments, the adjuvant may be one or more proteins and/ornucleic acid molecules that encode proteins selected from the groupconsisting of: CCL-20, IL-12, IL-15, IL-28, CTACK, TECK, MEC or RANTES.Examples of IL-12 constructs and sequences are disclosed in PCTapplication no. PCT/US1997/019502 and corresponding U.S. ApplicationSerial No. 08/956,865, and U.S. Provisional Application Serial No61/569600 filed Dec. 12, 2011, which are each incorporated herein byreference. Examples of IL-15 constructs and sequences are disclosed inPCT application no. PCT/US04/18962 and corresponding U.S. ApplicationSerial No. 10/560,650, and in PCT application no. PCT/US07/00886 andcorresponding U.S. Application Serial No. 12/160,766, and in PCTapplication no. PCT/US10/048827, which are each incorporated herein byreference. Examples of IL-28 constructs and sequences are disclosed inPCT application no. PCT/US09/039648 and corresponding U.S. ApplicationSerial No. 12/936,192, which are each incorporated herein by reference.Examples of RANTES and other constructs and sequences are disclosed inPCT application no. PCT/US1999/004332 and corresponding U.S. ApplicationSerial No. and 09/622452, which are each incorporated herein byreference. Other examples of RANTES constructs and sequences aredisclosed in PCT application no. PCT/US11/024098, which is incorporatedherein by reference. Examples of RANTES and other constructs andsequences are disclosed in PCT application no. PCT/US1999/004332 andcorresponding U.S. Application Serial No. 09/622452, which are eachincorporated herein by reference. Other examples of RANTES constructsand sequences are disclosed in PCT application no. PCT/US11/024098,which is incorporated herein by reference. Examples of chemokines CTACK,TECK and MEC constructs and sequences are disclosed in PCT applicationno. PCT/US2005/042231 and corresponding U.S. Application Serial No.11/719,646, which are each incorporated herein by reference. Examples ofOX40 and other immunomodulators are disclosed in U.S. Application SerialNo. 10/560,653, which is incorporated herein by reference. Examples ofDR5 and other immunomodulators are disclosed in U.S. Application SerialNo. 09/622452, which is incorporated herein by reference.

The immunogenic composition may further comprise a genetic vaccinefacilitator agent as described in U.S. Serial No. 021,579 filed Apr. 1,1994, which is fully incorporated by reference.

The immunogenic composition may comprise the consensus antigens andplasmids at quantities of from about 1 nanogram to 100 milligrams; about1 microgram to about 10 milligrams; or preferably about 0.1 microgram toabout 10 milligrams; or more preferably about 1 milligram to about 2milligram. In some preferred embodiments, pharmaceutical compositionsaccording to the present invention comprise about 5 nanogram to about1000 micrograms of DNA. In some preferred embodiments, thepharmaceutical compositions contain about 10 nanograms to about 800micrograms of DNA. In some preferred embodiments, the pharmaceuticalcompositions contain about 0.1 to about 500 micrograms of DNA. In somepreferred embodiments, the pharmaceutical compositions contain about 1to about 350 micrograms of DNA. In some preferred embodiments, thepharmaceutical compositions contain about 25 to about 250 micrograms,from about 100 to about 200 microgram, from about 1 nanogram to 100milligrams; from about 1 microgram to about 10 milligrams; from about0.1 microgram to about 10 milligrams; from about 1 milligram to about 2milligram, from about 5 nanogram to about 1000 micrograms, from about 10nanograms to about 800 micrograms, from about 0.1 to about 500micrograms, from about 1 to about 350 micrograms, from about 25 to about250 micrograms, from about 100 to about 200 microgram of the consensusantigen or plasmid thereof.

In some embodiments, pharmaceutical compositions according to thepresent invention comprise at least 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nanograms of a nucleic acidmolecule of the invention. In some embodiments, the pharmaceuticalcompositions can comprise at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 105, 110, 115, 120, 125,130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195,200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265,270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335,340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405,410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475,480, 485, 490, 495, 500, 605, 610, 615, 620, 625, 630, 635, 640, 645,650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715,720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785,790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855,860, 865, 870, 875, 880, 885, 890, 895. 900, 905, 910, 915, 920, 925,930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995 or1000 micrograms of a nucleic acid molecule of the invention. In someembodiments, the pharmaceutical composition can comprise at least 1.5,2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mgor more of a nucleic acid molecule of the invention.

In other embodiments, the pharmaceutical composition can comprise up toand including 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95 or 100 nanograms of a nucleic acid molecule of the invention.In some embodiments, the pharmaceutical composition can comprise up toand including 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95,100, 105, 110, 115, 120, 125, 130, 135, 140, 145,150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215,220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285,290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355,360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425,430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495,500, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665,670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735,740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805,810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875,880, 885, 890, 895. 900, 905, 910, 915, 920, 925, 930, 935, 940, 945,950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 micrograms ofa nucleic acid molecule of the invention. In some embodiments, thepharmaceutical composition can comprise up to and including 1.5, 2, 2.5,3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg of anucleic acid molecule of the invention.

The immunogenic composition may be formulated according to the mode ofadministration to be used. An injectable immunogenic compositionpharmaceutical composition may be sterile, pyrogen free and particulatefree. An isotonic formulation or solution may be used. Additives forisotonicity may include sodium chloride, dextrose, mannitol, sorbitol,and lactose. The immunogenic composition may comprise a vasoconstrictionagent. The isotonic solutions may include phosphate buffered saline.Immunogenic composition may further comprise stabilizers includinggelatin and albumin. The stabilizing may allow the formulation to bestable at room or ambient temperature for extended periods of time suchas LGS or polycations or polyanions to the immunogenic compositionformulation.

The immunogenic composition may be stable at room temperature (25° C.)for more than 1 week, in some embodiments for more than 2 weeks, in someembodiments for more than 3 weeks, in some embodiments for more than 4weeks, in some embodiments for more than 5 weeks, and in someembodiments for more than 6 weeks. In some embodiments, the vaccine isstable for more than one month, more than 2 months, more than 3 months,more than 4 months, more than 5 months, more than 6 months, more than 7months, more than 8 months, more than 9 months, more than 10 months,more than 11 months, or more than 12 months. In some embodiments, thevaccine is stable for more than 1 year, more than 2 years, more thanyears, or more than 5 years. In one embodiment, the immunogeniccomposition is stable under refrigeration (2-8° C.). Accordingly, in oneembodiment, the immunogenic composition does not require frozencold-chain. An immunogenic composition is stable if it retains itsbiological activity for a sufficient period to allow its intended use(e.g., to generate an immune response in a subject). For example, forimmunogenic compositions that are to be stored, shipped, etc., it may bedesired that the immunogenic compositions remain stable for months toyears.

Immune Response

The immunogenic composition can induce an immune response in the subjectadministered the composition. The induced immune response can bespecific for a MCV T antigen. The induced immune response can bereactive with a MCV T antigen related to the optimized consensus-encodedantigen. In various embodiments, related antigens include antigenshaving amino acid sequences having at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% homology to the amino acidsequence of the optimized consensus-encoded antigen. In variousembodiments, related antigens include antigens encoded by nucleotidesequences having at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% homology to the optimized consensus nucleotidesequences disclosed herein.

The immunogenic composition can induce a humoral immune response in thesubject administered the immunogenic composition. The induced humoralimmune response can be specific for a MCV T antigen. The induced humoralimmune response can be reactive with the MCV T antigen related to theoptimized consensus-encoded antigen. The humoral immune response can beinduced in the subject administered the immunogenic composition by about1.5-fold to about 16-fold, about 2-fold to about 12-fold, or about3-fold to about 10-fold. The humoral immune response can be induced inthe subject administered the immunogenic composition by at least about1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at leastabout 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, atleast about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold,at least about 6.0-fold, at least about 6.5-fold, at least about7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at leastabout 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, atleast about 10.0-fold, at least about 10.5-fold, at least about11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at leastabout 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, atleast about 14.0-fold, at least about 14.5-fold, at least about15.0-fold, at least about 15.5-fold, or at least about 16.0-fold ascompared to a subject not administered the immunogenic composition or asubject administered a non-optimized MCV T antigen.

The humoral immune response induced by the immunogenic composition caninclude an increased level of IgG antibodies associated with the subjectadministered the immunogenic composition as compared to a subject notadministered the immunogenic composition. These IgG antibodies can bespecific for the MCV T antigen genetically related to the optimizedconsensus antigen. These IgG antibodies can be reactive with the MCV Tantigen genetically related to the optimized consensus antigen. Thelevel of IgG antibody associated with the subject administered theimmunogenic composition can be increased by about 1.5-fold to about16-fold, about 2-fold to about 12-fold, or about 3-fold to about 10-foldas compared to the subject not administered the immunogenic composition.The level of IgG antibody associated with the subject administered theimmunogenic composition can be increased by at least about 1.5-fold, atleast about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold,at least about 3.5-fold, at least about 4.0-fold, at least about4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at leastabout 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, atleast about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold,at least about 9.0-fold, at least about 9.5-fold, at least about10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at leastabout 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, atleast about 13.0-fold, at least about 13.5-fold, at least about14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at leastabout 15.5-fold, or at least about 16.0-fold as compared to a subjectnot administered the immunogenic composition or a subject administered anon-optimized MCV T antigen.

The immunogenic composition can induce a cellular immune response in thesubject administered the immunogenic composition. The induced cellularimmune response can be specific for a MCV T antigen related to theoptimized consensus-encoded antigen. The induced cellular immuneresponse can be reactive to the MCV T antigen related to the optimizedconsensus-encoded antigen. The induced cellular immune response caninclude eliciting a CD8⁺ T cell response. The elicited CD8⁺ T cellresponse can be reactive with the MCV T antigen genetically related tothe optimized consensus antigen. The elicited CD8⁺ T cell response canbe polyfunctional. The induced cellular immune response can includeeliciting a CD8⁺ T cell response, in which the CD8⁺ T cells produceinterferon-gamma (IFN-y), tumor necrosis factor alpha (TNF-α),interleukin-2 (IL-2), or a combination of IFN-y and TNF-α.

The induced cellular immune response can include an increased CD8⁺ Tcell response associated with the subject administered the immunogeniccomposition as compared to the subject not administered the immunogeniccomposition. The CD8⁺ T cell response associated with the subjectadministered the immunogenic composition can be increased by about2-fold to about 30-fold, about 3-fold to about 25-fold, or about 4-foldto about 20-fold as compared to the subject not administered theimmunogenic composition. The CD8⁺ T cell response associated with thesubject administered the immunogenic composition can be increased by atleast about 1.5-fold, at least about 2.0-fold, at least about 3.0-fold,at least about 4.0-fold, at least about 5.0-fold, at least about6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at leastabout 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, atleast about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold,at least about 10.5-fold, at least about 11.0-fold, at least about11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at leastabout 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, atleast about 14.5-fold, at least about 15.0-fold, at least about16.0-fold, at least about 17.0-fold, at least about 18.0-fold, at leastabout 19.0-fold, at least about 20.0-fold, at least about 21.0-fold, atleast about 22.0-fold, at least about 23.0-fold, at least about24.0-fold, at least about 25.0-fold, at least about 26.0-fold, at leastabout 27.0-fold, at least about 28.0-fold, at least about 29.0-fold, orat least about 30.0-fold as compared to a subject not administered theimmunogenic composition or a subject administered a non-optimized MCV Tantigen.

The induced cellular immune response can include an increased frequencyof CD107a/IFNy/T-bet triple-positive CD8 T cells that are reactiveagainst the MCV T antigen. The frequency of CD107a/IFNy/T-bettriple-positive CD8 T cells associated with the subject administered theimmunogenic composition can be increased by at least about 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold,19-fold, or 20-fold as compared to a subject not administered theimmunogenic composition or a subject administered a non-optimized MCV Tantigen.

The induced cellular immune response can include an increased frequencyof CD107a/IFNy double-positive CD8 T cells that are reactive against theMCV T antigen. The frequency of CD107a/IFNy double-positive CD8 T cellsassociated with the subject administered the immunogenic composition canbe increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, or 14-foldas compared to a subject not administered the immunogenic composition ora subject administered a non-optimized MCV T antigen.

The cellular immune response induced by the immunogenic composition caninclude eliciting a CD4⁺ T cell response. The elicited CD4⁺ T cellresponse can be reactive with the MCV T antigen genetically related tothe optimized consensus antigen. The elicited CD4⁺ T cell response canbe polyfunctional. The induced cellular immune response can includeeliciting a CD4⁺ T cell response, in which the CD4⁺ T cells produceIFN-y, TNF-α, IL-2, or a combination of IFN-y and TNF-α.

The induced cellular immune response can include an increased frequencyof CD4⁺ T cells that produce IFN-y. The frequency of CD4⁺IFN-γ⁺ T cellsassociated with the subject administered the immunogenic composition canbe increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold,15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold as compared to asubject not administered the immunogenic composition or a subjectadministered a non-optimized MCV T antigen.

The induced cellular immune response can include an increased frequencyof CD4⁺ T cells that produce TNF-α. The frequency of CD4^(÷)TNF-α^(÷) Tcells associated with the subject administered the immunogeniccomposition can be increased by at least about 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold,13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold,21-fold, or 22-fold as compared to a subject not administered theimmunogenic composition or a subject administered a non-optimized MCV Tantigen.

The induced cellular immune response can include an increased frequencyof CD4⁺ T cells that produce both IFN-y and TNF-α. The frequency ofCD4⁺IFN-γ⁺TNF-α⁺ associated with the subject administered theimmunogenic composition can be increased by at least about 2-fold,2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5-fold,6.0-fold, 6.5-fold, 7.0-fold, 7.5-fold, 8.0-fold, 8.5-fold, 9.0-fold,9.5-fold, 10.0-fold, 10.5-fold, 11.0-fold, 11.5-fold, 12.0-fold,12.5-fold, 13.0-fold, 13.5-fold, 14.0-fold, 14.5-fold, 15.0-fold,15.5-fold, 16.0-fold, 16.5-fold, 17.0-fold, 17.5-fold, 18.0-fold,18.5-fold, 19.0-fold, 19.5-fold, 20.0-fold, 21-fold, 22-fold, 23-fold24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold,32-fold, 33-fold, 34-fold, or 35-fold as compared to a subject notadministered the immunogenic composition or a subject administered anon-optimized MCV T antigen.

The immunogenic composition can further induce an immune response whenadministered to different tissues such as the muscle or skin. Theimmunogenic composition can further induce an immune response whenadministered via electroporation, or injection, or subcutaneously, orintramuscularly.

Vector

The nucleotide construct described above can be placed in one or morevectors. The one or more vectors can contain an origin of replication.The one or more vectors can be a plasmid, bacteriophage, bacterialartificial chromosome or yeast artificial chromosome. The one or morevectors can be either a self-replication extra chromosomal vector, or avector which integrates into a host genome.

Vectors include, but are not limited to, plasmids, expression vectors,recombinant viruses, any form of recombinant “naked DNA” vector, and thelike. A “vector” comprises a nucleic acid which can infect, transfect,transiently or permanently transduce a cell. It will be recognized thata vector can be a naked nucleic acid, or a nucleic acid complexed withprotein or lipid. The vector optionally comprises viral or bacterialnucleic acids and/or proteins, and/or membranes (e.g., a cell membrane,a viral lipid envelope, etc.). Vectors include, but are not limited toreplicons (e.g., RNA replicons, bacteriophages) to which fragments ofDNA may be attached and become replicated. Vectors thus include, but arenot limited to RNA, autonomous self-replicating circular or linear DNAor RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No.5,217,879), and include both the expression and non-expression plasmids.Where a recombinant microorganism or cell culture is described ashosting an “expression vector” this includes both extra-chromosomalcircular and linear DNA and DNA that has been incorporated into the hostchromosome(s). Where a vector is being maintained by a host cell, thevector may either be stably replicated by the cells during mitosis as anautonomous structure, or is incorporated within the host’s genome.

The one or more vectors can be an expression construct, which isgenerally a plasmid that is used to introduce a specific gene into atarget cell. Once the expression vector is inside the cell, the proteinthat is encoded by the gene is produced by the cellular-transcriptionand translation machinery ribosomal complexes. The plasmid is frequentlyengineered to contain regulatory sequences that act as enhancer andpromoter regions and lead to efficient transcription of the gene carriedon the expression vector. The vectors of the present invention expresslarge amounts of stable messenger RNA, and therefore proteins.

The vectors may have expression signals such as a strong promoter, astrong termination codon, adjustment of the distance between thepromoter and the cloned gene, and the insertion of a transcriptiontermination sequence and a PTIS (portable translation initiationsequence).

Expression Vector

The one or more vectors can be a circular plasmid or a linear nucleicacid. The circular plasmid and linear nucleic acid are capable ofdirecting expression of a particular nucleotide sequence in anappropriate subject cell. The one or more vectors comprising therecombinant nucleic acid construct may be chimeric, meaning that atleast one of its components is heterologous with respect to at least oneof its other components.

Plasmid

The one or more vectors can be a plasmid. The plasmid may be useful fortransfecting cells with the recombinant nucleic acid construct. Theplasmid may be useful for introducing the recombinant nucleic acidconstruct into the subject. The plasmid may also comprise a regulatorysequence, which may be well suited for gene expression in a cell intowhich the plasmid is administered.

The plasmid may also comprise a mammalian origin of replication in orderto maintain the plasmid extrachromosomally and produce multiple copiesof the plasmid in a cell. The plasmid may be pVAX1, pCEP4 or pREP4 fromInvitrogen (San Diego, CA), which may comprise the Epstein Barr virusorigin of replication and nuclear antigen EBNA-1 coding region, whichmay produce high copy episomal replication without integration. Thebackbone of the plasmid may be pAV0242. The plasmid may be a replicationdefective adenovirus type 5 (Ad5) plasmid.

The plasmid may be pSE420 (Invitrogen, San Diego, Calif.), which may beused for protein production in Escherichia coli (E.coli). The plasmidmay also be pYES2 (Invitrogen, San Diego, Calif.), which may be used forprotein production in Saccharomyces cerevisiae strains of yeast. Theplasmid may also be of the MAXBAC™ complete baculovirus expressionsystem (Invitrogen, San Diego, Calif.), which may be used for proteinproduction in insect cells. The plasmid may also be pcDNAI or pcDNA3(Invitrogen, San Diego, Calif.), which may be used for proteinproduction in mammalian cells such as Chinese hamster ovary (CHO) cells.

RNA

In one embodiment, the nucleic acid is an RNA molecule. In oneembodiment, the RNA molecule is transcribed from a DNA sequencedescribed herein. For example, in some embodiments, the RNA molecule isencoded by a DNA sequence at least 90% homologous to one of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, or a variant thereof or a fragmentthereof. In another embodiment, the nucleotide sequence comprises an RNAsequence transcribed by a DNA sequence encoding a polypeptide sequenceat least 90% homologous to one of SEQ ID NO:2, SEQ ID NO:4 or SEQ IDNO:6 or a variant thereof or a fragment thereof. Accordingly, in oneembodiment, the invention provides an RNA molecule encoding one or moreof the MCV T antigens. The RNA may be plus-stranded. Accordingly, insome embodiments, the RNA molecule can be translated by cells withoutneeding any intervening replication steps such as reverse transcription.A RNA molecule useful with the invention may have a 5′ cap (e.g. a7-methylguanosine). This cap can enhance in vivo translation of the RNA.The 5′ nucleotide of a RNA molecule useful with the invention may have a5′ triphosphate group. In a capped RNA this may be linked to a7-methylguanosine via a 5′-to-5′ bridge. A RNA molecule may have a 3′poly-A tail. It may also include a poly-A polymerase recognitionsequence (e.g. AAUAAA) near its 3′ end. A RNA molecule useful with theinvention may be single-stranded. A RNA molecule useful with theinvention may comprise synthetic RNA. In some embodiments, the RNAmolecule is a naked RNA molecule. In one embodiment, the RNA molecule iscomprised within a vector.

In one embodiment, the RNA has 5′ and 3′ UTRs. In one embodiment, the 5′UTR is between zero and 3000 nucleotides in length. The length of 5′ and3′ UTR sequences to be added to the coding region can be altered bydifferent methods, including, but not limited to, designing primers forPCR that anneal to different regions of the UTRs. Using this approach,one of ordinary skill in the art can modify the 5′ and 3′ UTR lengthsrequired to achieve optimal translation efficiency followingtransfection of the transcribed RNA.

The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the gene of interest. Alternatively, UTR sequences that are notendogenous to the gene of interest can be added by incorporating the UTRsequences into the forward and reverse primers or by any othermodifications of the template. The use of UTR sequences that are notendogenous to the gene of interest can be useful for modifying thestability and/or translation efficiency of the RNA. For example, it isknown that AU-rich elements in 3′ UTR sequences can decrease thestability of RNA. Therefore, 3′ UTRs can be selected or designed toincrease the stability of the transcribed RNA based on properties ofUTRs that are well known in the art.

In one embodiment, the 5′ UTR can contain the Kozak sequence of theendogenous gene. Alternatively, when a 5′ UTR that is not endogenous tothe gene of interest is being added by PCR as described above, aconsensus Kozak sequence can be redesigned by adding the 5′ UTRsequence. Kozak sequences can increase the efficiency of translation ofsome RNA transcripts, but does not appear to be required for all RNAs toenable efficient translation. The requirement for Kozak sequences formany RNAs is known in the art. In other embodiments, the 5′ UTR can bederived from an RNA virus whose RNA genome is stable in cells. In otherembodiments, various nucleotide analogues can be used in the 3′ or 5′UTR to impede exonuclease degradation of the RNA.

In one embodiment, the RNA has both a cap on the 5′ end and a 3′ poly(A)tail which determine ribosome binding, initiation of translation andstability of RNA in the cell.

In one embodiment, the RNA is a nucleoside-modified RNA.Nucleoside-modified RNA have particular advantages over non-modifiedRNA, including for example, increased stability, low or absent innateimmunogenicity, and enhanced translation.

Circular and Linear Vector

The one or more vectors may be circular plasmid, which may transform atarget cell by integration into the cellular genome or existextrachromosomally (e.g., autonomous replicating plasmid with an originof replication). The vector can be pVAX, pcDNA3.0, or provax, or anyother expression vector capable of expressing the heavy chainpolypeptide and/or light chain polypeptide encoded by the recombinantnucleic acid construct.

Also provided herein is a linear nucleic acid, or linear expressioncassette (“LEC”), that is capable of being efficiently delivered to asubject via electroporation and expressing the heavy chain polypeptideand/or light chain polypeptide encoded by the recombinant nucleic acidconstruct. The LEC may be any linear DNA devoid of any phosphatebackbone. The LEC may not contain any antibiotic resistance genes and/ora phosphate backbone. The LEC may not contain other nucleotide sequencesunrelated to the desired gene expression.

The LEC may be derived from any plasmid capable of being linearized. Theplasmid may be capable of expressing the heavy chain polypeptide and/orlight chain polypeptide encoded by the recombinant nucleic acidconstruct. The plasmid can be pNP (Puerto Rico/34) or pM2 (NewCaledonia/99). The plasmid may be WLV009, pVAX, pcDNA3.0, or provax, orany other expression vector capable of expressing the heavy chainpolypeptide and/or light chain polypeptide encoded by the recombinantnucleic acid construct.

The LEC can be pcrM2. The LEC can be pcrNP. pcrNP and pcrMR can bederived from pNP (Puerto Rico/34) and pM2 (New Caledonia/99),respectively.

Viral Vectors

In one embodiment, viral vectors are provided herein which are capableof delivering a nucleic acid of the invention to a cell. The expressionvector may be provided to a cell in the form of a viral vector. Viralvector technology is well known in the art and is described, forexample, in Sambrook et al. (2001), and in Ausubel et al. (1997), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers. (See, e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Method of Preparing the Vector

Provided herein is a method for preparing the one or more vectors inwhich the recombinant nucleic acid construct has been placed. After thefinal subcloning step, the vector can be used to inoculate a cellculture in a large scale fermentation tank, using known methods in theart.

In other embodiments, after the final subcloning step, the vector can beused with one or more electroporation (EP) devices. The EP devices aredescribed below in more detail.

The one or more vectors can be formulated or manufactured using acombination of known devices and techniques, but preferably they aremanufactured using a plasmid manufacturing technique that is describedin a licensed, co-pending U.S. provisional application U.S. Serial No.60/939,792, which was filed on May 23, 2007. In some examples, the DNAplasmids described herein can be formulated at concentrations greaterthan or equal to 10 mg/mL. The manufacturing techniques also include orincorporate various devices and protocols that are commonly known tothose of ordinary skill in the art, in addition to those described inU.S. Serial No. 60/939792, including those described in a licensedpatent, U.S. Pat. No. 7,238,522, which issued on Jul. 3, 2007. Theabove-referenced application and patent, U.S. Serial No. 60/939,792 andU.S. Pat. No. 7,238,522, respectively, are hereby incorporated in theirentirety.

Multiple Vectors

The immunogenic composition may comprise a plurality of copies of asingle nucleic acid molecule such a single plasmid, or a plurality ofcopies of two or more different nucleic acid molecules such as two ormore different plasmids. For example an immunogenic composition maycomprise plurality of two, three, four, five, six, seven, eight, nine orten or more different nucleic acid molecules. Such compositions maycomprise plurality of two, three, four, five, six, or more differentplasmids.

Immunogenic compositions may comprise nucleic acid molecules, such asplasmids, that collectively contain coding sequence for a MCV T antigen.Immunogenic compositions may comprise nucleic acid molecules, such asplasmids, that collectively contain coding sequence for multipleantigens. In one embodiment, the antigens are a MCV T antigen and one ormore additional cancer antigen. Immunogenic compositions may comprisenucleic acid molecules, such as plasmids, that collectively containcoding sequence for one or more MCV T antigen and one or more cancerantigen.

Cancer Antigens

The immunogenic composition can comprise one or more cancer antigenssuch as WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2/neu, Idiotype, MAGEA3, p53 (non-mutant), NY-ESO-1, PSMA, GD2, CEA, MelanA/MART1,Ras-mutant, gp100, p53 mutant, Proteinase 3 (PR1), Bcr-abl, Tyrosinase,Survivin, PSA, hTERT, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG, NA17, PAX3,ALK, Androgen Receptor, Cyclin B1, Polysialic Acid, MYCN, TRP-2, RhoC,GD3, Fucosyl GM1, Mesothelin, PSCA, MAGE A1, sLe(a), CYP1B1, PLAC1, GM3ganglioside, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn,Carbonic anhydrase IX, PAX5, OY-TES1, Sperm Protein 17, LCK, HMWMAA,Sperm fibrous sheath proteins, AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie2, Page4, VEGFR2, MAD-CT-1 (protamine 2), MAD-CT-2, and FOS-relatedantigen 1 to treat or prevent a tumor associated pathology. Theimmunogenic composition can further combine one or more cancer antigensWT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2/neu, Idiotype, MAGE A3, p53(non-mutant), NY-ESO-1, PSMA, GD2, CEA, MelanA/MART1, Ras-mutant, gp100,p53 mutant, Proteinase 3 (PR1), Bcr-abl, Tyrosinase, Survivin, PSA,hTERT, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG, NA17, PAX3, ALK, AndrogenReceptor, Cyclin B1, Polysialic Acid, MYCN, TRP-2, RhoC, GD3, FucosylGM1, Mesothelin, PSCA, MAGE A1, sLe(a), CYP1B1, PLAC1, GM3 ganglioside,BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonicanhydrase IX, PAX5, OY-TES1, Sperm Protein 17, LCK, HMWMAA, Spermfibrous sheath proteins, AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 2,Page4, VEGFR2, MAD-CT-1 (protamine 2), MAD-CT-2, and FOS-related antigenwith an optimized consensus encoded MCV T antigen for treating orpreventing a tumor associated pathology. Other combinations of cancerantigens may also be applied for treating or preventing a tumorassociated pathology.

Methods

Provided herein are methods of treating, protecting against, and/orpreventing a MCV associated disease in a subject in need thereof byadministering one or more immunogenic composition described herein tothe subject. Administration of the immunogenic composition to thesubject can induce or elicit an immune response in the subject. Theinduced immune response can be used to treat, prevent, and/or protectagainst disease, for example, MCV infection or MCC associated with MCVinfection.

Provided herein is a method for delivering the immunogenic compositionfor providing genetic constructs and proteins of the consensus antigenwhich comprise epitopes that make them particular effective against MCVor MCC, against which an immune response can be induced. The method ofdelivering the immunogenic composition or vaccination may be provided toinduce a therapeutic and prophylactic immune response. The vaccinationprocess may generate in the mammal an immune response against MCV orMCC. The immunogenic composition may be delivered to an individual tomodulate the activity of the mammal’s immune system and enhance theimmune response. The delivery of the immunogenic composition may be thetransfection of the consensus antigen as a nucleic acid molecule that isexpressed in the cell and delivered to the surface of the cell uponwhich the immune system recognized and induces a cellular, humoral, orcellular and humoral response. The delivery of the immunogeniccomposition may be used to induce or elicit and immune response inmammals against MCV or MCC by administering to the mammals theimmunogenic composition as discussed above.

Upon delivery of the immunogenic composition and plasmid into the cellsof the mammal, the transfected cells will express and secrete consensusantigens for each of the plasmids injected from the immunogeniccomposition. These proteins will be recognized as foreign by the immunesystem and antibodies will be made against them. These antibodies willbe maintained by the immune system and allow for an effective responseto subsequent infections by MCV.

The immunogenic composition may be administered to a mammal to elicit animmune response in a mammal. The mammal may be human, primate, non-humanprimate, cow, cattle, sheep, goat, antelope, bison, water buffalo,bison, bovids, deer, hedgehogs, elephants, llama, alpaca, mice, rats,and chicken.

The induced immune response can include an induced humoral immuneresponse and/or an induced cellular immune response. The humoral immuneresponse can be induced by about 1.5-fold to about 16-fold, about 2-foldto about 12-fold, or about 3-fold to about 10-fold. The induced cellularimmune response can include a CD8⁺ T cell response, which is induced byabout 2-fold to about 30-fold, about 3-fold to about25-fold, or about4-fold to about 20-fold.

The immunogenic composition dose can be between 1 µg to 10 mg activecomponent/kg body weight/time, and can be 20 µg to 10 mg component/kgbody weight/time. The immunogenic composition can be administered every1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number ofimmunogenic composition doses for effective treatment can be 1, 2, 3, 4,5, 6, 7, 8, 9, or 10.

The immunogenic composition can be formulated in accordance withstandard techniques well known to those skilled in the pharmaceuticalart. Such compositions can be administered in dosages and by techniqueswell known to those skilled in the medical arts taking intoconsideration such factors as the age, sex, weight, and condition of theparticular subject, and the route of administration.

The immunogenic composition can be administered prophylactically ortherapeutically. In prophylactic administration, the immunogeniccompositions can be administered in an amount sufficient to induce animmune response. In therapeutic applications, the immunogeniccompositions are administered to a subject in need thereof in an amountsufficient to elicit a therapeutic effect. An amount adequate toaccomplish this is defined as “therapeutically effective dose.” Amountseffective for this use will depend on, e.g., the particular compositionof the immunogenic composition regimen administered, the manner ofadministration, the stage and severity of the disease, the general stateof health of the subject, and the judgment of the prescribing physician.

The immunogenic composition can be administered by methods well known inthe art as described in Donnelly et al. (Ann. Rev. Immunol. 15:617-648(1997)); Felgner et al. (U.S. Pat. No. 5,580,859, issued Dec. 3, 1996);Felgner (U.S. Pat. No. 5,703,055, issued Dec. 30, 1997); and Carson etal. (U.S. Pat. No. 5,679,647, issued Oct. 21, 1997), the contents of allof which are incorporated herein by reference in their entirety. The DNAof the immunogenic composition can be complexed to particles or beadsthat can be administered to an individual, for example, using a vaccinegun. One skilled in the art would know that the choice of apharmaceutically acceptable carrier, including a physiologicallyacceptable compound, depends, for example, on the route ofadministration of the expression vector.

The immunogenic composition can be delivered via a variety of routes.Typical delivery routes include parenteral administration, e.g.,intradermal, intramuscular or subcutaneous delivery. Other routesinclude oral administration, intranasal, and intravaginal routes. Forthe DNA of the immunogenic composition in particular, the immunogeniccomposition can be delivered to the interstitial spaces of tissues of anindividual (Felgner et al., U.S. Pat. Nos. 5,580,859 and 5,703,055, thecontents of all of which are incorporated herein by reference in theirentirety). The immunogenic composition can also be administered tomuscle, or can be administered via intradermal or subcutaneousinjections, or transdermally, such as by iontophoresis. Epidermaladministration of the immunogenic composition can also be employed.Epidermal administration can involve mechanically or chemicallyirritating the outermost layer of epidermis to stimulate an immuneresponse to the irritant (Carson et al., U.S. Pat. No. 5,679,647, thecontents of which are incorporated herein by reference in its entirety).

The immunogenic composition can also be formulated for administrationvia the nasal passages. Formulations suitable for nasal administration,wherein the carrier is a solid, can include a coarse powder having aparticle size, for example, in the range of about 10 to about 500microns which is administered in the manner in which snuff is taken,i.e., by rapid inhalation through the nasal passage from a container ofthe powder held close up to the nose. The formulation can be a nasalspray, nasal drops, or by aerosol administration by nebulizer. Theformulation can include aqueous or oily solutions of the immunogeniccomposition.

The immunogenic composition can be a liquid preparation such as asuspension, syrup or elixir. The immunogenic composition can also be apreparation for parenteral, subcutaneous, intradermal, intramuscular orintravenous administration (e.g., injectable administration), such as asterile suspension or emulsion.

The immunogenic composition can be incorporated into liposomes,microspheres or other polymer matrices (Felgner et al., U.S. Pat. No.5,703,055; Gregoriadis, Liposome Technology, Vols. Ito III (2nd ed.1993), the contents of which are incorporated herein by reference intheir entirety). Liposomes can consist of phospholipids or other lipids,and can be nontoxic, physiologically acceptable and metabolizablecarriers that are relatively simple to make and administer.

Method of Cancer Treatment With the Vaccine

The vaccine can be used to generate or elicit an immune response in amammal that is reactive or directed to a cancer or tumor (e.g., MCC) ofthe mammal or subject in need thereof. The elicited immune response canprevent cancer or tumor growth.

The elicited immune response can prevent and/or reduce metastasis ofcancerous or tumor cells. Accordingly, the vaccine can be used in amethod that treats and/or prevents cancer or tumors in the mammal orsubject administered the vaccine.

In some embodiments, the administered vaccine can mediate clearance orprevent growth of tumor cells by inducing (1) humoral immunity via Bcell responses to generate antibodies that block monocytechemoattractant protein-1 (MCP-1) production, thereby retarding myeloidderived suppressor cells (MDSCs) and suppressing tumor growth; (2)increase cytotoxic T lymphocyte such as CD8+ (CTL) to attack and killtumor cells; (3) increase T helper cell responses; (4) and increaseinflammatory responses via IFN-y and TFN-α or preferably all of theaforementioned.

In some embodiments, the immune response can generate a humoral immuneresponse and/or an antigen-specific cytotoxic T lymphocyte (CTL)response that does not cause damage to or inflammation of varioustissues or systems (e.g., brain or neurological system, etc.) in thesubject administered the vaccine.

In some embodiments, the administered vaccine can increase tumor freesurvival, reduce tumor mass, increase tumor survival, or a combinationthereof in the subject. The administered vaccine can increase tumor freesurvival by 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %,32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,and 60% or more in the subject. The administered vaccine can reducetumor mass by 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, and 70% or morein the subject after immunization. The administered vaccine can preventand block increases in monocyte chemoattractant protein 1 (MCP-1), acytokine secreted by myeloid derived suppressor cells, in the subject.In some embodiments, the administered vaccine can prevent and blockincreases in MCP-1 within the cancerous or tumor tissue in the subject,thereby reducing vascularization of the cancerous or tumor tissue in thesubject.

The administered vaccine can increase tumor survival by 20%, 21%, 22%,23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, and 70% or more in the subject. In someembodiments, the vaccine can be administered to the periphery (asdescribed in more detail below) to establish an antigen-specific immuneresponse targeting the cancerous or tumor cells or tissue to clear oreliminate the cancer or tumor expressing the one or more MCV T antigenswithout damaging or causing illness or death in the subject administeredthe vaccine.

The administered vaccine can increase a cellular immune response in thesubject by about 50-fold to about 6000-fold, about 50-fold to about5500-fold, about 50-fold to about 5000-fold, about 50-fold to about4500-fold, about 100-fold to about 6000-fold, about 150-fold to about6000-fold, about 200-fold to about 6000-fold, about 250-fold to about6000-fold, or about 300-fold to about 6000-fold. In some embodiments,the administered vaccine can increase the cellular immune response inthe subject by about 50-fold, 100-fold, 150-fold, 200-fold, 250-fold,300-fold, 350-fold, 400-fold, 450-fold, 500-fold, 550-fold, 600-fold,650-fold, 700-fold, 750-fold, 800-fold, 850-fold, 900-fold, 950-fold,1000-fold, 1100-fold, 1200-fold, 1300-fold, 1400-fold, 1500-fold,1600-fold, 1700-fold, 1800-fold, 1900-fold, 2000-fold, 2100-fold,2200-fold, 2300-fold, 2400-fold, 2500-fold, 2600-fold, 2700-fold,2800-fold, 2900-fold, 3000-fold, 3100-fold, 3200-fold, 3300-fold,3400-fold, 3500-fold, 3600-fold, 3700-fold, 3800-fold, 3900-fold,4000-fold, 4100-fold, 4200-fold, 4300-fold, 4400-fold, 4500-fold,4600-fold, 4700-fold, 4800-fold, 4900-fold, 5000-fold, 5100-fold,5200-fold, 5300-fold, 5400-fold, 5500-fold, 5600-fold, 5700-fold,5800-fold, 5900-fold, or 6000-fold.

The administered vaccine can increase interferon gamma (IFN-y) levels inthe subject by about 50-fold to about 6000-fold, about 50-fold to about5500-fold, about 50-fold to about 5000-fold, about 50-fold to about4500-fold, about 100-fold to about 6000-fold, about 150-fold to about6000-fold, about 200-fold to about 6000-fold, about 250-fold to about6000-fold, or about 300-fold to about 6000-fold. In some embodiments,the administered vaccine can increase IFN-y levels in the subject byabout 50-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold,350-fold, 400-fold, 450-fold, 500-fold, 550-fold, 600-fold, 650-fold,700-fold, 750-fold, 800-fold, 850-fold, 900-fold, 950-fold, 1000-fold,1100-fold, 1200-fold, 1300-fold, 1400-fold, 1500-fold, 1600-fold,1700-fold, 1800-fold, 1900-fold, 2000-fold, 2100-fold, 2200-fold,2300-fold, 2400-fold, 2500-fold, 2600-fold, 2700-fold, 2800-fold,2900-fold, 3000-fold, 3100-fold, 3200-fold, 3300-fold, 3400-fold,3500-fold, 3600-fold, 3700-fold, 3800-fold, 3900-fold, 4000-fold,4100-fold, 4200-fold, 4300-fold, 4400-fold, 4500-fold, 4600-fold,4700-fold, 4800-fold, 4900-fold, 5000-fold, 5100-fold, 5200-fold,5300-fold, 5400-fold, 5500-fold, 5600-fold, 5700-fold, 5800-fold,5900-fold, or 6000-fold.

The vaccine dose can be between 1 µg to 10 mg active component/kg bodyweight/time and can be 20 µg to 10 mg component/kg body weight/time. Thevaccine can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,or 31 days. The number of vaccine doses for effective treatment can be1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

Combinational Therapies With Checkpoint Inhibitors

The present invention is also directed to a method of increasing animmune response in a mammal using the vaccine as described above incombination with one or more checkpoint inhibitor. In one embodiment,the vaccine as described above can comprise the MCV T antigen and anantibody to a checkpoint protein. “Checkpoint inhibitor” as used hereinincludes inhibitors or molecules that block immune checkpoints ascommonly understood in the field of cancer immunotherapy. More commonlythe checkpoint inhibitors are antibodies that block the immunecheckpoint proteins. Immune checkpoint proteins include, but are notlimited to, PD1, PDL1, PDL2, CTLA-4, LAG3, TIM3, B7-H3, BTLA, VISTA,CD40, CEACAM1, CD80, CD86, OX40, CD27, GITR, DNAM-1, TIGIT, TMIGD2 andDC-SIGN. Some examples of known checkpoint inhibitors include, but arenot limited to, ipilimumab, pembrolizumab, nivolumab, pidilizumab,avelumab and others.

The combination can be in a single formulation or can be separate andadministered in sequence (either MCV T antigen first and then checkpointinhibitor, or checkpoint inhibitor first and then MCV T antigen). Insome embodiments, the MCV T antigen can be administered to the subjectabout 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes,10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes,40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 0.25 hours,0.5 hours, 0.75 hours, 1 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 hours, 60hours, 72 hours, 84 hours, 96 hours, 1 day, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29days, 30 days, 31 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 7 weeks, or 8 weeks before the checkpoint inhibitor isadministered to the subject. In other embodiments, the checkpointinhibitor can be administered to the subject about 30 seconds, 1 minute,2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50minutes, 55 minutes, 60 minutes, 0.25 hours, 0.5 hours, 0.75 hours, 1hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks beforethe MCV T antigen is administered to the subject.

The combination of the MCV T antigen and checkpoint inhibitor inducesthe immune system more efficiently than a vaccine comprising the MCV Tantigen alone. This more efficient immune response provides increasedefficacy in the treatment and/or prevention of a particular cancer.

In some embodiments, the immune response can be increased by about0.5-fold to about 15-fold, about 0.5-fold to about 10-fold, or about0.5-fold to about 8-fold. Alternatively, the immune response in thesubject administered the vaccine can be increased by at least about0.5-fold, at least about 1.0-fold, at least about 1.5-fold, at leastabout 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, atleast about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold,at least about 5.0-fold, at least about 5.5-fold, at least about6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at leastabout 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, atleast about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold,at least about 10.5-fold, at least about 11.0-fold, at least about11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at leastabout 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, atleast about 14.5-fold, or at least about 15.0-fold.

In still other alternative embodiments, the immune response in thesubject administered the vaccine can be increased about 50% to about1500%, about 50% to about 1000%, or about 50% to about 800%. In otherembodiments, the immune response in the subject administered the vaccinecan be increased by at least about 50%, at least about 100%, at leastabout 150%, at least about 200%, at least about 250%, at least about300%, at least about 350%, at least about 400%, at least about 450%, atleast about 500%, at least about 550%, at least about 600%, at leastabout 650%, at least about 700%, at least about 750%, at least about800%, at least about 850%, at least about 900%, at least about 950%, atleast about 1000%, at least about 1050%, at least about 1100%, at leastabout 1150%, at least about 1200%, at least about 1250%, at least about1300%, at least about 1350%, at least about 1450%, or at least about1500%.

The vaccine dose can be between 1 µg to 10 mg active component/kg bodyweight/time, and can be 20 µg to 10 mg component/kg body weight/time.The vaccine can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, or 31 days. The number of vaccine doses for effective treatment canbe 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

Merkel Cell Carcinoma

The vaccine can be used to generate or elicit an immune response in amammal that is reactive or directed to a Merkel Cell Carcinoma (MCC) inthe mammal or subject in need thereof. The elicited immune response canprevent MCC growth. The elicited immune response can reduce MCC growth.The elicited immune response can prevent and/or reduce metastasis ofcancerous or tumor cells from a MCC. Accordingly, the vaccine can beused in a method that treats and/or prevents MCC in the mammal orsubject administered the vaccine.

In some embodiments, the administered vaccine can mediate clearance orprevent growth of MCC by inducing (1) humoral immunity via B cellresponses to generate antibodies that target an MCV T antigen expressedby MCC cells; (2) increase cytotoxic T lymphocyte such as CD8+ (CTL) toattack and kill MCC cells; (3) increase T helper cell responses; and (4)increase inflammatory responses via IFN-y and TFN-α or all of theaforementioned.

In some embodiments, the administered vaccine can increase MCC freesurvival, reduce MCC mass, increase MCC survival, or a combinationthereof in the subject. The administered vaccine can increase MCC freesurvival by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,42%, 43%, 44%, and 45% or more in the subject. The administered vaccinecan reduce MCC mass by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, and 60% or more in the subject afterimmunization. The administered vaccine can prevent and block increasesin monocyte chemoattractant protein 1 (MCP-1), a cytokine secreted bymyeloid derived suppressor cells, in the subject. In some embodiments,the administered vaccine can prevent and block increases in MCP-1 withinthe MCC tissue in the subject, thereby reducing vascularization of theMCC tissue in the subject. The administered vaccine can increase MCCsurvival by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, and 60% or more in the subject.

Combination Treatments

The immunogenic composition may be administered in combination withother proteins and/or genes encoding CCL20, α-interferon, γ-interferon,platelet derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermalgrowth factor (EGF), cutaneous T cell-attracting chemokine (CTACK),epithelial thymus-expressed chemokine (TECK), mucosae-associatedepithelial chemokine (MEC), IL-12, IL-15 including IL-15 having thesignal sequence deleted and optionally including the different signalpeptide such as the IgE signal peptide, MHC, CD80, CD86, IL-28, IL-1,IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIP-lα, MIP-1β, IL-8,RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1,LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3,M-CSF, G-CSF, mutant forms of IL-18, CD40, CD40L, vascular growthfactor, fibroblast growth factor, IL-7, nerve growth factor, vascularendothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1,DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2,DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88,IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1, JNK, interferon responsegenes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4,RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B,NKG2C, NKG2E, NKG2F, TAP1, TAP2 and functional fragments thereof orcombinations thereof. In some embodiments, the immunogenic compositionis administered in combination with one or more of the following nucleicacid molecules and/or proteins: nucleic acid molecules selected from thegroup consisting of nucleic acid molecules comprising coding sequencethat encode one or more of CCL20, IL-12, IL-15, IL-28, CTACK, TECK, MECand RANTES or functional fragments thereof, and proteins selected fromthe group consisting of: CCL02, IL-12 protein, IL-15 protein, IL-28protein, CTACK protein, TECK protein, MEC protein or RANTES protein orfunctional fragments thereof.

The immunogenic composition may be administered by different routesincluding orally, parenterally, sublingually, transdermally, rectally,transmucosally, topically, via inhalation, via buccal administration,intrapleurally, intravenous, intraarterial, intraperitoneal,subcutaneous, intramuscular, intranasal, intrathecal, and intraarticularor combinations thereof. For veterinary use, the composition may beadministered as a suitably acceptable formulation in accordance withnormal veterinary practice. The veterinarian can readily determine thedosing regimen and route of administration that is most appropriate fora particular animal. The immunogenic composition may be administered bytraditional syringes, needleless injection devices, “microprojectilebombardment gone guns”, or other physical methods such aselectroporation (“EP”), “hydrodynamic method”, or ultrasound.

The plasmid of the immunogenic composition may be delivered to themammal by several well-known technologies including DNA injection (alsoreferred to as DNA vaccination) with and without in vivoelectroporation, liposome mediated, nanoparticle facilitated,recombinant vectors such as recombinant adenovirus, recombinantadenovirus associated virus and recombinant vaccinia. The consensusantigen may be delivered via DNA injection and along with in vivoelectroporation.

Electroporation

Administration of the immunogenic composition via electroporation may beaccomplished using electroporation devices that can be configured todeliver to a desired tissue of a mammal a pulse of energy effective tocause reversible pores to form in cell membranes, and preferable thepulse of energy is a constant current similar to a preset current inputby a user. The electroporation device may comprise an electroporationcomponent and an electrode assembly or handle assembly. Theelectroporation component may include and incorporate one or more of thevarious elements of the electroporation devices, including: controller,current waveform generator, impedance tester, waveform logger, inputelement, status reporting element, communication port, memory component,power source, and power switch. The electroporation may be accomplishedusing an in vivo electroporation device, for example CELLECTRA EP system(Inovio Pharmaceuticals, Plymouth Meeting, PA) or Elgen electroporator(Inovio Pharmaceuticals, Plymouth Meeting, PA) to facilitatetransfection of cells by the plasmid.

The electroporation component may function as one element of theelectroporation devices, and the other elements are separate elements(or components) in communication with the electroporation component. Theelectroporation component may function as more than one element of theelectroporation devices, which may be in communication with still otherelements of the electroporation devices separate from theelectroporation component. The elements of the electroporation devicesexisting as parts of one electromechanical or mechanical device may notlimited as the elements can function as one device or as separateelements in communication with one another. The electroporationcomponent may be capable of delivering the pulse of energy that producesthe constant current in the desired tissue, and includes a feedbackmechanism. The electrode assembly may include an electrode array havinga plurality of electrodes in a spatial arrangement, wherein theelectrode assembly receives the pulse of energy from the electroporationcomponent and delivers same to the desired tissue through theelectrodes. At least one of the plurality of electrodes is neutralduring delivery of the pulse of energy and measures impedance in thedesired tissue and communicates the impedance to the electroporationcomponent. The feedback mechanism may receive the measured impedance andcan adjust the pulse of energy delivered by the electroporationcomponent to maintain the constant current.

A plurality of electrodes may deliver the pulse of energy in adecentralized pattern. The plurality of electrodes may deliver the pulseof energy in the decentralized pattern through the control of theelectrodes under a programmed sequence, and the programmed sequence isinput by a user to the electroporation component. The programmedsequence may comprise a plurality of pulses delivered in sequence,wherein each pulse of the plurality of pulses is delivered by at leasttwo active electrodes with one neutral electrode that measuresimpedance, and wherein a subsequent pulse of the plurality of pulses isdelivered by a different one of at least two active electrodes with oneneutral electrode that measures impedance.

The feedback mechanism may be performed by either hardware or software.The feedback mechanism may be performed by an analog closed-loopcircuit. The feedback occurs every 50 µs, 20 µs, 10 µs or 1 µs, but ispreferably a real-time feedback or instantaneous (i.e., substantiallyinstantaneous as determined by available techniques for determiningresponse time). The neutral electrode may measure the impedance in thedesired tissue and communicates the impedance to the feedback mechanism,and the feedback mechanism responds to the impedance and adjusts thepulse of energy to maintain the constant current at a value similar tothe preset current. The feedback mechanism may maintain the constantcurrent continuously and instantaneously during the delivery of thepulse of energy.

Examples of electroporation devices and electroporation methods that mayfacilitate delivery of the immunogenic compositions of the presentinvention, include those described in U.S. Pat. No. 7,245,963 byDraghia-Akli, et al., U.S. Pat. Pub. 2005/0052630 submitted by Smith, etal., the contents of which are hereby incorporated by reference in theirentirety. Other electroporation devices and electroporation methods thatmay be used for facilitating delivery of the immunogenic compositionsinclude those provided in co-pending and co-owned U.S. Pat. Application,Serial No. 11/874072, filed Oct. 17, 2007, which claims the benefitunder 35 USC 119(e) to U.S. Provisional Applications Ser. Nos.60/852,149, filed Oct. 17, 2006, and 60/978,982, filed Oct. 10, 2007,all of which are hereby incorporated in their entirety.

U.S. Pat. No. 7,245,963 by Draghia-Akli, et al. describes modularelectrode systems and their use for facilitating the introduction of abiomolecule into cells of a selected tissue in a body or plant. Themodular electrode systems may comprise a plurality of needle electrodes;a hypodermic needle; an electrical connector that provides a conductivelink from a programmable constant-current pulse controller to theplurality of needle electrodes; and a power source. An operator cangrasp the plurality of needle electrodes that are mounted on a supportstructure and firmly insert them into the selected tissue in a body orplant. The biomolecules are then delivered via the hypodermic needleinto the selected tissue. The programmable constant-current pulsecontroller is activated and constant-current electrical pulse is appliedto the plurality of needle electrodes. The applied constant-currentelectrical pulse facilitates the introduction of the biomolecule intothe cell between the plurality of electrodes. The entire content of U.S.Pat. No. 7,245,963 is hereby incorporated by reference.

U.S. Pat. Pub. 2005/0052630 submitted by Smith, et al. describes anelectroporation device which may be used to effectively facilitate theintroduction of a biomolecule into cells of a selected tissue in a bodyor plant. The electroporation device comprises an electro-kinetic device(“EKD device”) whose operation is specified by software or firmware. TheEKD device produces a series of programmable constant-current pulsepatterns between electrodes in an array based on user control and inputof the pulse parameters, and allows the storage and acquisition ofcurrent waveform data. The electroporation device also comprises areplaceable electrode disk having an array of needle electrodes, acentral injection channel for an injection needle, and a removable guidedisk. The entire content of U.S. Pat. Pub. 2005/0052630 is herebyincorporated by reference.

The electrode arrays and methods described in U.S. Pat. No. 7,245,963and U.S. Pat. Pub. 2005/0052630 may be adapted for deep penetration intonot only tissues such as muscle, but also other tissues or organs.Because of the configuration of the electrode array, the injectionneedle (to deliver the biomolecule of choice) is also insertedcompletely into the target organ, and the injection is administeredperpendicular to the target issue, in the area that is pre-delineated bythe electrodes The electrodes described in U.S. Pat. No. 7,245,963 andU.S. Pat. Pub. 2005/005263 are preferably 20 mm long and 21 gauge.

Additionally, contemplated in some embodiments that incorporateelectroporation devices and uses thereof, there are electroporationdevices that are those described in the following patents: U.S. Pat.5,273,525 issued Dec. 28, 1993, U.S. Pats. 6,110,161 issued Aug. 29,2000, 6,261,281 issued Jul. 17, 2001, and 6,958,060 issued Oct. 25,2005, and U.S. Pat. 6,939,862 issued Sep. 6, 2005. Furthermore, patentscovering subject matter provided in U.S. Pat. 6,697,669 issued Feb. 24,2004, which concerns delivery of DNA using any of a variety of devices,and U.S. Pat. 7,328,064 issued Feb. 5, 2008, drawn to method ofinjecting DNA are contemplated herein. The above-patents areincorporated by reference in their entirety.

Generation of Antigens In Vitro and Ex Vivo

In one embodiment, the optimized consensus MCV T antigen is generated invitro or ex vivo. For example, in one embodiment, a nucleic acidencoding an optimized consensus MCV T antigen can be introduced andexpressed in an in vitro or ex vivo cell.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2012,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). A preferred method for the introduction of a polynucleotideinto a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

EXAMPLES

The present invention is further illustrated in the following Example.It should be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, various modifications of the invention in addition tothose shown and described herein will be apparent to those skilled inthe art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Example 1: Nucleic Acid Vaccine Targeting Merkel Cell Polyomavirus

A nucleic acid vaccine targeting Merkel Cell Polyomavirus (MCV) Tantigens has been developed (FIG. 1 and FIG. 2 ). Optimized syntheticconsensus MCV T antigen sequences representing the large T antigen(LTAg) and small t antigen (STAg) were individually cloned intomammalian expression-plasmid DNA (FIG. 3 ) and delivered to mice viaintramuscular electroporation (FIG. 4A). Following immunization, DNAvaccine constructs generated robust antibody and T-cell responsesagainst MCV T antigen peptides (FIG. 4B through FIG. 15 ).

FIG. 4B, FIG. 7 , FIG. 12 and FIG. 14 demonstrate that the LTAg vaccineis highly immunogenic in C57B⅙ and CD-1 outbred mice. FIG. 8 throughFIG. 10 demonstrate that LTAg vaccination results in robust,polyfunctional CD4 and CD8 T cells and cytotoxic CD8 T cells.

FIG. 4B and FIG. 15 demonstrate that STAg vaccine is immunogenic inC57B⅙ and CD-1 mice. FIG. 15 demonstrates that both CD4 and CD8responses were detected for IFNγ/TNFα for CD-1 mice.

FIG. 11 demonstrates that both vaccines generate humoral response inC57B⅙ mice.

Example 2: Sequences

SEQ ID NO:1: Nucleotide sequence encoding modified synthetic consensusMCV LTAg

ATGGACCTGGTGCTGAACAGGAAGGAGAGAGAGGCCCTGTGCAAGCTGCTGGAGATCGCCCCCAACTGTTACGGCAATATCCCTCTGATGAAGGCCGCCTTCAAGCGGAGCTGCCTGAAGCACCACCCCAACAAGGGCGGCAACCCTGTGATCATGATGGAGCTGAATACCCTGTGGTCCAAGTTTCAGCAGAATATCCACAAGCTGCGGTCCGATTTCTCTATGTTTGACGAGGTGGATGAGGCCCCTATCTACGGCACCACCAAGTTCAAGGAGTGGTGGCGCTCCGGCGGCTTCTCTTTTGGCAAGGCCTACGAGTACGGCCCTAACCCACACGGCACCAATAGCAGGTCCAGAAAGCCAAGCTCCAACGCCAGCAGGGGAGCACCATCCGGATCTAGCCCACCTCACAGCCAGTCCTCTAGCTCCGGCTACGGCTCTTTTAGCGCCTCCCAGGCCTCTGACAGCCAGTCCAGAGGCCCCGATATCCCACCCGAGCACCACGAGGAGCCTACCTCTAGCTCCGGCTCTAGCTCCCGGGAGGAGACAACCAACAGCGGCAGGGAGTCTAGCACCCCAAACGGCACCTCCGTGCCAAGGAATTCCTCTAGGACCGACGGAACCGCCGAGGACCTGTTCTGCGATAAGTCCCTGAGCTCCCCTGAGCCTCCATCTAGCTCCGAGGAGCCAGAGGAGCCCCCTTCTAGCAGGTCCTCTCCCAGACAGCCACCAAGCTCCTCTGCCGAGGAGGCAAGCTCCTCTCAGTTCACCGACGAGGAGTACAGGAGCTCCTCTTTTACCACCCCTAAGACCCCTCCACCCTTCTCCCGGAAGCGCAAGTTTGGAGGCTCTAGGAGCTCCGCCTCTAGCGCCTCCTCTGCCAGCTTCACCTCCACCCCTCCAAAGCCCAAGAAGAACAGAGAGACACCCGTGCCTACCGACTTTCCTATCGACCTGAGCGATTACCTGTCCCACGCCGTGTACTCTAATAAGACCGTGAGCTGTTTCGCCATCTACACCACCAGCGACAAGGCCATCGAGCTGTACGATAAGATCGAGAAGTTCAAGGTGGACTTCAAGTCCAGGCACGCATGCGAGCTGGGATGTATCCTGCTGTTCATCACCCTGTCCAAGCACCGCGTGTCTGCCATCAAGAACTTCTGCAGCACCTTTTGTACCATCTCCTTTCTGATCTGCAAGGGCGTGAATAAGATGCCTGAGATGTACAACAACCTGTGCAAGCCCCCTTACAAGCTGCTGCAGGAGAACAAGCCACTGCTGAATTACGAGTTCCAGGAGAAGGAGAAGGAGGCCAGCTGCAACTGGAATCTGGTGGCCGAGTTCGCCTGTGAGTACGAGCTGGACGATCACTTTATCATCCTGGCCCACTACCTGGACTTCGCCAAGCCATTTCCCTGCCAGAAGTGTGAGAACAGGTCTAGACTGAAGCCACACAAGGCCCACGAGGCCCACCACTCCAATGCCAAGCTGTTTTACGAGTCTAAGAGCCAGAAGACCATCTGCCAGCAGGCAGCAGACACCGTGCTGGCAAAGAGGAGACTGGAGATGCTGGAGATGACCAGGACCGAGATGCTGTGCAAGAAGTTCAAGAAGCACCTGGAGCGGCTGCGCGACCTGGATACCATCGATCTGCTGTACTACATGGGCGGCGTGGCCTGGTACTGCTGTCTGTTCGAGGAGTTTGAGAAGAAGCTGCAGAAGATCATCCAGCTGCTGACCGAGAACATCCCAAAGTACAGAAATATCTGGTTCAAGGGCCCCATCAACTCTGGCAAGACCAGCTTCGCCGCCGCCCTGATCGACCTGCTGGAGGGCAAGGCCCTGAACATCAATTGCCCTAGCGATAAGCTGCCATTCGAGCTGGGCTGTGCCCTGGACAAGTTCATGGTGGTGTTTGAGGATGTGAAGGGCCAGAACTCCCTGAATAAGGACCTGCAGCCCGGCCAGGGCATCAACAATCTGGATAACCTGCGGGACCACCTGGATGGAGCAGTGGCCGTGAGCCTGGAGAAGAAGCACGTGAACAAGAAGCACCAGATCTTCCCACCCTGCATCGTGACCGCCAATGACTACTTTATCCCAAAGACCCTGATCGCCCGCTTCTCTTACACCCTGCACTTTAGCCCCAAGGCCAACCTGAGGGACAGCCTGGATCAGAATATGGAGATCAGAAAGAGGCGCATCCTGCAGTCCGGAACCACCCTGCTGCTGTGCCTGATCTGGTGTCTGCCTGACACCACCTTCAAGCCATGCCTGCAGGAGGAGATCAAGAACTGGAAGCAGATCCTGCAGTCTGAGATCAGCTACGGCAAGTTTTGTCAGATGATCGAGAACGTGGAGGCCGGCCAGGACCCCCTGCTGAATATCCTGATCGAGGAGGAGGGCCCAGAGGAGACAGAGGAGACACAGGACTCCGGCACCTTCTCTCA G

SEQ ID NO:2: Amino acid sequence of modified synthetic consensus MCVLTAg

MDLVLNRKEREALCKLLEIAPNCYGNIPLMKAAFKRSCLKHHPNKGGNPVIMMELNTLWSKFQQNIHKLRSDFSMFDEVDEAPIYGTTKFKEWWRSGGFSFGKAYEYGPNPHGTNSRSRKPSSNASRGAPSGSSPPHSQSSSSGYGSFSASQASDSQSRGPDIPPEHHEEPTSSSGSSSREETTNSGRESSTPNGTSVPRNSSRTDGTAEDLFCDKSLSSPEPPSSSEEPEEPPSSRSSPRQPPSSSAEEASSSQFTDEEYRSSSFTTPKTPPPFSRKRKFGGSRSSASSASSASFTSTPPKPKKNRETPVPTDFPIDLSDYLSHAVYSNKTVSCFAIYTTSDKAIELYDKIEKFKVDFKSRHACELGCILLFITLSKHRVSAIKNFCSTFCTISFLICKGVNKMPEMYNNLCKPPYKLLQENKPLLNYEFQEKEKEASCNWNLVAEFACEYELDDHFIILAHYLDFAKPFPCQKCENRSRLKPHKAHEAHHSNAKLFYESKSQKTICQQAADTVLAKRRLEMLEMTRTEMLCKKFKKHLERLRDLDTIDLLYYMGGVAWYCCLFEEFEKKLQKIIQLLTENIPKYRNIWFKGPINSGKTSFAAALIDLLEGKALNINCPSDKLPFELGCALDKFMVVFEDVKGQNSLNKDLQPGQGINNLDNLRDHLDGAVAVSLEKKHVNKKHQIFPPCIVTANDYFIPKTLIARFSYTLHFSPKANLRDSLDQNMEIRKRRILQSGTTLLLCLIWCLPDTTFKPCLQEEIKNWKQILQSEISYGKFCQMIENVEAGQDPLLNILIEE EGPEETEETQDSGTFSQ

SEQ ID NO:3: Nucleotide sequence encoding modified synthetic consensusMCV STAg

ATGGACCTGGTGCTGAACCGAAAGGAGAGGGAGGCCCTGTGCAAGCTGCTGGAGATCGCCCCTAACTGTTACGGCAATATCCCACTGATGAAGGCCGCCTTCAAGAGGTCTTGCCTGAAGCACCACCCAAACAAGGGCGGCAATCCCGTGATCATGATGGAGCTGAACACCCTGTGGAGCAAGTTTCAGCAGAATATCCACAAGCTGCGGAGCGACTTCTCCATGTTTGATGAGGTGAGCACCAAGTTCCCCTGGGAGGAGTACGGAACAGCAGCAGCAGCAGCACAGTCCGGCTATAACGCCAGGTTTTGCAGAGGCCCTGGCTGTATGCTGAAGCAGCTGCGGGACTCCAAGTGCGCCTGTATCTCTTGCAAGCTGAGCCGCCAGCACTGTTCTCTGAAGACCCTGAAGCAGAAGAATTGCGCCACATGGGGCGAGTGCTTCTGTTATCAGTGTTTTATCCTGTGGTTCGGCTTTCCCCCTACATGGGAGTCCTTCGATTGGTGGCAGAAAACCCTGGAAGAAACCGACTACTGTCTGCTGCATCTGC ATCTGTTC

SEQ ID NO:4: Amino acid sequence of modified synthetic consensus MCVSTAg

MDLVLNRKEREALCKLLEIAPNCYGNIPLMKAAFKRSCLKHHPNKGGNPVIMMELNTLWSKFQQNIHKLRSDFSMFDEVSTKFPWEEYGTAAAAAQSGYNARFCRGPGCMLKQLRDSKCACISCKLSRQHCSLKTLKQKNCATWGECFCYQCFILWFGFPPTWESFDWWQKTLEETDYCLLHLHLF

SEQ ID NO:5: Nucleotide sequence encoding modified synthetic consensusLTAg and STAg linked with a furin cleavage site

ATGGACCTGGTGCTGAACAGGAAGGAGAGAGAGGCCCTGTGCAAGCTGCTGGAGATCGCCCCCAACTGTTACGGCAATATCCCTCTGATGAAGGCCGCCTTCAAGCGGAGCTGCCTGAAGCACCACCCCAACAAGGGCGGCAACCCTGTGATCATGATGGAGCTGAATACCCTGTGGTCCAAGTTTCAGCAGAATATCCACAAGCTGCGGTCCGATTTCTCTATGTTTGACGAGGTGGATGAGGCCCCTATCTACGGCACCACCAAGTTCAAGGAGTGGTGGCGCTCCGGCGGCTTCTCTTTTGGCAAGGCCTACGAGTACGGCCCTAACCCACACGGCACCAATAGCAGGTCCAGAAAGCCAAGCTCCAACGCCAGCAGGGGAGCACCATCCGGATCTAGCCCACCTCACAGCCAGTCCTCTAGCTCCGGCTACGGCTCTTTTAGCGCCTCCCAGGCCTCTGACAGCCAGTCCAGAGGCCCCGATATCCCACCCGAGCACCACGAGGAGCCTACCTCTAGCTCCGGCTCTAGCTCCCGGGAGGAGACAACCAACAGCGGCAGGGAGTCTAGCACCCCAAACGGCACCTCCGTGCCAAGGAATTCCTCTAGGACCGACGGAACCGCCGAGGACCTGTTCTGCGATAAGTCCCTGAGCTCCCCTGAGCCTCCATCTAGCTCCGAGGAGCCAGAGGAGCCCCCTTCTAGCAGGTCCTCTCCCAGACAGCCACCAAGCTCCTCTGCCGAGGAGGCAAGCTCCTCTCAGTTCACCGACGAGGAGTACAGGAGCTCCTCTTTTACCACCCCTAAGACCCCTCCACCCTTCTCCCGGAAGCGCAAGTTTGGAGGCTCTAGGAGCTCCGCCTCTAGCGCCTCCTCTGCCAGCTTCACCTCCACCCCTCCAAAGCCCAAGAAGAACAGAGAGACACCCGTGCCTACCGACTTTCCTATCGACCTGAGCGATTACCTGTCCCACGCCGTGTACTCTAATAAGACCGTGAGCTGTTTCGCCATCTACACCACCAGCGACAAGGCCATCGAGCTGTACGATAAGATCGAGAAGTTCAAGGTGGACTTCAAGTCCAGGCACGCATGCGAGCTGGGATGTATCCTGCTGTTCATCACCCTGTCCAAGCACCGCGTGTCTGCCATCAAGAACTTCTGCAGCACCTTTTGTACCATCTCCTTTCTGATCTGCAAGGGCGTGAATAAGATGCCTGAGATGTACAACAACCTGTGCAAGCCCCCTTACAAGCTGCTGCAGGAGAACAAGCCACTGCTGAATTACGAGTTCCAGGAGAAGGAGAAGGAGGCCAGCTGCAACTGGAATCTGGTGGCCGAGTTCGCCTGTGAGTACGAGCTGGACGATCACTTTATCATCCTGGCCCACTACCTGGACTTCGCCAAGCCATTTCCCTGCCAGAAGTGTGAGAACAGGTCTAGACTGAAGCCACACAAGGCCCACGAGGCCCACCACTCCAATGCCAAGCTGTTTTACGAGTCTAAGAGCCAGAAGACCATCTGCCAGCAGGCAGCAGACACCGTGCTGGCAAAGAGGAGACTGGAGATGCTGGAGATGACCAGGACCGAGATGCTGTGCAAGAAGTTCAAGAAGCACCTGGAGCGGCTGCGCGACCTGGATACCATCGATCTGCTGTACTACATGGGCGGCGTGGCCTGGTACTGCTGTCTGTTCGAGGAGTTTGAGAAGAAGCTGCAGAAGATCATCCAGCTGCTGACCGAGAACATCCCAAAGTACAGAAATATCTGGTTCAAGGGCCCCATCAACTCTGGCAAGACCAGCTTCGCCGCCGCCCTGATCGACCTGCTGGAGGGCAAGGCCCTGAACATCAATTGCCCTAGCGATAAGCTGCCATTCGAGCTGGGCTGTGCCCTGGACAAGTTCATGGTGGTGTTTGAGGATGTGAAGGGCCAGAACTCCCTGAATAAGGACCTGCAGCCCGGCCAGGGCATCAACAATCTGGATAACCTGCGGGACCACCTGGATGGAGCAGTGGCCGTGAGCCTGGAGAAGAAGCACGTGAACAAGAAGCACCAGATCTTCCCACCCTGCATCGTGACCGCCAATGACTACTTTATCCCAAAGACCCTGATCGCCCGCTTCTCTTACACCCTGCACTTTAGCCCCAAGGCCAACCTGAGGGACAGCCTGGATCAGAATATGGAGATCAGAAAGAGGCGCATCCTGCAGTCCGGAACCACCCTGCTGCTGTGCCTGATCTGGTGTCTGCCTGACACCACCTTCAAGCCATGCCTGCAGGAGGAGATCAAGAACTGGAAGCAGATCCTGCAGTCTGAGATCAGCTACGGCAAGTTTTGTCAGATGATCGAGAACGTGGAGGCCGGCCAGGACCCCCTGCTGAATATCCTGATCGAGGAGGAGGGCCCAGAGGAGACAGAGGAGACACAGGACTCCGGCACCTTCTCTCAGAGAGGCCGCAAAAGGAGGTCTGATCTGGTGCTGAATCGGAAAGAGAGAGAAGCCCTGTGCAAACTGCTGGAAATCGCCCCAAACTGTTACGGCAACATCCCCCTGATGAAGGCCGCCTTCAAGAGGTCTTGCCTGAAGCACCACCCAAACAAGGGCGGCAATCCCGTGATCATGATGGAGCTGAACACCCTGTGGAGCAAGTTTCAGCAGAATATCCACAAGCTGCGGAGCGACTTCTCCATGTTTGATGAGGTGAGCACCAAGTTCCCTTGGGAGGAGTACGGAACAGCAGCAGCAGCAGCACAGTCCGGCTATAACGCCAGGTTTTGCAGAGGCCCAGGCTGTATGCTGAAGCAGCTGCGGGACTCCAAGTGCGCCTGTATCTCTTGCAAGCTGAGCCGCCAGCACTGTTCTCTGAAGACCCTGAAGCAGAAGAATTGCGCCACATGGGGCGAGTGCTTCTGTTATCAGTGTTTTATCCTGTGGTTCGGCTTTCCCCCTACATGGGAGTCCTTCGATTGGTGGCAGAAAACCCTGGAGGAAACTGATTACTGTCTGCTGCACCTGCACCTGTTC

SEQ ID NO:6: Amino acid sequence of modified synthetic consensus LTAgand STAg linked with a furin cleavage site.

MDLVLNRKEREALCKLLEIAPNCYGNIPLMKAAFKRSCLKHHPNKGGNPVIMMELNTLWSKFQQNIHKLRSDFSMFDEVDEAPIYGTTKFKEWWRSGGFSFGKAYEYGPNPHGTNSRSRKPSSNASRGAPSGSSPPHSQSSSSGYGSFSASQASDSQSRGPDIPPEHHEEPTSSSGSSSREETTNSGRESSTPNGTSVPRNSSRTDGTAEDLFCDKSLSSPEPPSSSEEPEEPPSSRSSPRQPPSSSAEEASSSQFTDEEYRSSSFTTPKTPPPFSRKRKFGGSRSSASSASSASFTSTPPKPKKNRETPVPTDFPIDLSDYLSHAVYSNKTVSCFAIYTTSDKAIELYDKIEKFKVDFKSRHACELGCILLFITLSKHRVSAIKNFCSTFCTISFLICKGVNKMPEMYNNLCKPPYKLLQENKPLLNYEFQEKEKEASCNWNLVAEFACEYELDDHFIILAHYLDFAKPFPCQKCENRSRLKPHKAHEAHHSNAKLFYESKSQKTICQQAADTVLAKRRLEMLEMTRTEMLCKKFKKHLERLRDLDTIDLLYYMGGVAWYCCLFEEFEKKLQKIIQLLTENIPKYRNIWFKGPINSGKTSFAAALIDLLEGKALNINCPSDKLPFELGCALDKFMVVFEDVKGQNSLNKDLQPGQGINNLDNLRDHLDGAVAVSLEKKHVNKKHQIFPPCIVTANDYFIPKTLIARFSYTLHFSPKANLRDSLDQNMEIRKRRILQSGTTLLLCLIWCLPDTTFKPCLQEEIKNWKQILQSEISYGKFCQMIENVEAGQDPLLNILIEEEGPEETEETQDSGTFSQRGRKRRSDLVLNRKEREALCKLLEIAPNCYGNIPLMKAAFKRSCLKHHPNKGGNPVIMMELNTLWSKFQQNIHKLRSDFSMFDEVSTKFPWEEYGTAAAAAQSGYNARFCRGPGCMLKQLRDSKCACISCKLSRQHCSLKTLKQKNCATWGECFCYQCFILWFGFPPTWESFDWWQKTLEETD YCLLHLHLF

SEQ ID NO:7: Amino acid sequence of IgE leader sequence

MDWTWILFLVAAATRVHS

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative and are not to be taken aslimitations upon the scope of the invention, which is defined solely bythe appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art. Such changes and modifications,including without limitation those relating to the chemical structures,substituents, derivatives, intermediates, syntheses, compositions,formulations, or methods of use of the invention, may be made withoutdeparting from the spirit and scope thereof.

1-30. (canceled)
 31. A nucleic acid molecule encoding at least onemodified Merkel Cell Polyomavirus (MCV) T antigen, wherein the T antigencomprises at least one mutation that disrupts at least one oncogenicfeature of a native MCV T antigen.
 32. The nucleic acid molecule ofclaim 31, wherein the at least one oncogenic feature is selected fromthe group consisting of CR1 binding, DnaJ binding, phophatasepp2A-binding binding, Rb binding, ATPase activity, helicase activity,chaperone protein binding, hVam6p binding, Fbxw7 binding, originbinding, and transformation.
 33. The nucleic acid molecule of claim 31,wherein at least one mutation is a mutation at an amino acid selectedfrom the group consisting of D44, W209, E216, L142, L91, K92, D93, Y94and M95.
 34. The nucleic acid molecule of claim 31, wherein at least onemutation is selected from the group consisting of a D44N mutation, aW209A, an E216K mutation, an L142A mutation, an L91A mutation, a K92Amutation, a D93A mutation, a Y94A mutation and a M95A mutation.
 35. Thenucleic acid molecule of claim 31, wherein the MCV T antigen is selectedfrom the group consisting of a large T antigen (LTAg), a small t antigen(STAg), and a combination thereof.
 36. The nucleic acid molecule ofclaim 31, encoding at least one peptide comprising an amino acidsequence selected from the group consisting of a) an amino acid sequencehaving at least about 90% identity over an entire length of the aminoacid sequence selected from the group consisting of SEQ ID NO:2, SEQ IDNO:4 and SEQ ID NO:6, b) an immunogenic fragment comprising at leastabout 90% identity over at least 60% of the amino acid sequence selectedfrom the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6,c) the amino acid sequence selected from the group consisting of SEQ IDNO:2, SEQ ID NO:4 and SEQ ID NO:6, and d) an immunogenic fragmentcomprising at least 60% of the amino acid sequence selected from thegroup consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6.
 37. Thenucleic acid molecule of claim 31, wherein the nucleic acid molecule isselected from the group consisting of a DNA molecule and an RNAmolecule.
 38. The nucleic acid molecule of claim 31, wherein the nucleicacid molecule comprises at least one nucleotide sequence selected fromthe group consisting of a) a nucleotide sequence having at least about80% identity over an entire length of a nucleotide sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO:3 and SEQ ID NO:5,b) an immunogenic fragment of a nucleotide sequence having at leastabout 80% identity over at least 60% of the nucleotide sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO:3 and SEQ ID NO:5,and c) an immunogenic fragment comprising at least 60% of the nucleotidesequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3and SEQ ID NO:5.
 39. The nucleic acid molecule of claim 31, wherein theencoded peptide is operably linked to at least one regulatory sequenceselected from the group consisting of a start codon, an IgE leadersequence and a stop codon.
 40. The nucleic acid molecule of claim 39,wherein the nucleic acid molecule encodes at least one peptidecomprising an amino acid sequence selected from the group consisting ofa) an amino acid sequence having at least about 90% identity over anentire length of the amino acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6, b) animmunogenic fragment comprising at least about 90% identity over atleast 60% of the amino acid sequence selected from the group consistingof SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6, c) the amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQID NO:6, and d) an immunogenic fragment comprising at least 60% of theamino acid sequence selected from the group consisting of SEQ ID NO:2,SEQ ID NO:4 and SEQ ID NO:6, operably linked to an amino acid sequenceas set forth in SEQ ID NO:7.
 41. The nucleic acid molecule of claim 39,wherein the nucleic acid molecule comprises a nucleotide sequenceselected from the group consisting of a) a nucleotide sequence having atleast about 80% identity over an entire length of a nucleotide sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO:4 and SEQID NO:5, b) an immunogenic fragment of a nucleotide sequence having atleast about 80% identity over at least 60% of the nucleotide sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3 and SEQID NO:5,and c) an immunogenic fragment comprising at least 60% of thenucleotide sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO:3 and SEQ ID NO:5, operably linked to an nucleotide sequenceencoding SEQ ID NO:7.
 42. The nucleic acid molecule of claim 31, whereinthe nucleic acid molecule comprises an expression vector.
 43. Thenucleic acid molecule of claim 31, wherein the nucleic acid moleculecomprises a viral particle.
 44. An immunogenic composition comprising atleast one nucleic acid molecule of claim
 31. 45. The immunogeniccomposition of claim 44, further comprising at least one selected fromthe group consisting of a pharmaceutically acceptable excipient and anadjuvant.
 46. A method of treating or preventing a MCV associatedpathology in subject in need thereof, the method comprisingadministering to the subject a peptide comprising an amino acid sequenceselected from the group consisting of a) an amino acid sequence havingat least about 90% identity over an entire length of the amino acidsequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4and SEQ ID NO:6, b) an immunogenic fragment comprising at least about90% identity over at least 60% of the amino acid sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6, c) theamino acid sequence selected from the group consisting of SEQ ID NO:2,SEQ ID NO:4 and SEQ ID NO:6, and d) an immunogenic fragment comprisingat least 60% of the amino acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6.
 47. A method oftreating or preventing a MCV associated pathology in subject in needthereof, the method comprising administering to the subject animmunogenic composition comprising a peptide comprising an amino acidsequence selected from the group consisting of a) an amino acid sequencehaving at least about 90% identity over an entire length of the aminoacid sequence selected from the group consisting of SEQ ID NO:2, SEQ IDNO:4 and SEQ ID NO:6, b) an immunogenic fragment comprising at leastabout 90% identity over at least 60% of the amino acid sequence selectedfrom the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID N0:6,c) the amino acid sequence selected from the group consisting of SEQ IDNO:2, SEQ ID NO: 4 and SEQ ID NO:6, and d) an immunogenic fragmentcomprising at least 60% of the amino acid sequence selected from thegroup consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6.
 48. Amethod of inducing an immune response against a MCV T antigen in asubject in need thereof, the method comprising administering animmunogenic composition of claim 44 to the subject.
 49. A method oftreating or preventing a MCV associated pathology in subject in needthereof, the method comprising administering an immunogenic compositionof claim 31 to the subject.
 50. The method of claim 49, wherein the MCVassociated pathology is at least one of MCV infection and Merkel CellCarcinoma.